Methods and compositions for increasing replication capacity of an influenza virus

ABSTRACT

In certain aspects, the present invention provides methods for increasing the replication capacity of influenza viruses in hens&#39; eggs and/or cell culture, recombinant and/or reassortant influenza viruses with increased replication capacity, and immunogenic and vaccine compositions comprising such recombinant and/or reassortant influenza viruses. In other aspects, the invention further provides nucleic acids encoding influenza genes associated with increased replication capacity, expression vectors comprising the nucleic acids of the invention, methods for making influenza viruses with increased replication capacity, and kits useful for practice of the methods.

1. CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119 (e) of U.S.Provisional Application No. 60/832,553 filed Jul. 21, 2006, thedisclosure of which is incorporated herein in its entirety for allpurposes.

2. FIELD OF THE INVENTION

In certain aspects, the present invention provides methods forincreasing the replication capacity of influenza viruses in hens' eggsand/or cell culture, recombinant and/or reassortant influenza viruseswith increased replication capacity, and immunogenic and vaccinecompositions comprising such recombinant and/or reassortant influenzaviruses. In other aspects, the invention further provides nucleic acidsencoding influenza genes associated with increased replication capacity,expression vectors comprising the nucleic acids of the invention,methods for making influenza viruses with increased replicationcapacity, and kits useful for practice of the methods.

3. BACKGROUND

Influenza viruses are made up of an internal ribonucleoprotein corecontaining a segmented single-stranded RNA genome and an outerlipoprotein envelope lined by a matrix protein. Influenza A and Bviruses each contain eight segments of single stranded RNA with negativepolarity. The influenza A genome encodes at least eleven polypeptides.Segments 1-3 encode the three polypeptides, making up the viralRNA-dependent RNA polymerase. Segment 1 encodes the polymerase complexprotein PB2. The remaining polymerase proteins PB1 and PA are encoded bysegment 2 and segment 3, respectively. In addition, segment 1 of someinfluenza A strains encodes a small protein, PB1-F2, produced from analternative reading frame within the PB1 coding region. Segment 4encodes the hemagglutinin (HA) surface glycoprotein involved in cellattachment and entry during infection. Segment 5 encodes thenucleocapsid nucleoprotein (NP) polypeptide, the major structuralcomponent associated with viral RNA. Segment 6 encodes a neuraminidase(NA) envelope glycoprotein. Segment 7 encodes two matrix proteins,designated M1 and M2, which are translated from differentially splicedmRNAs. Segment 8 encodes NS1 and NS2 (NEP), two nonstructural proteins,which are translated from alternatively spliced mRNA variants.

The eight genome segments of influenza B encode 11 proteins. The threelargest genes code for components of the RNA polymerase, PB1, PB2 andPA. Segment 4 encodes the HA protein. Segment 5 encodes NP. Segment 6encodes the NA protein and the NB protein. Both proteins, NB and NA, aretranslated from overlapping reading frames of a biscistronic mRNA.Segment 7 of influenza B also encodes two proteins: M1 and BM2. Thesmallest segment encodes two products: NS1 is translated from the fulllength RNA, while NS2 is translated from a spliced mRNA variant.

Vaccines capable of producing a protective immune response specific forinfluenza viruses have been produced for over 50 years. Vaccines can becharacterized as whole virus vaccines, split virus vaccines, surfaceantigen vaccines and live attenuated virus vaccines. While appropriateformulations of any of these vaccine types is able to produce a systemicimmune response, live attenuated virus vaccines are also able tostimulate local mucosal immunity in the respiratory tract.

To date, all commercially available influenza vaccines in the UnitedStates have been propagated in embryonated hens' eggs. Althoughinfluenza virus generally grows well in hens' eggs, some influenzavaccine strains, such as the prototype A/Fujian/411/02 strain thatcirculated during the 2003-04 season, do not replicate well inembryonated hens' eggs, and have to be isolated by cell culture in acostly and time consuming procedure.

The ability of certain influenza virus strains to replicate to hightiter in embryonated hens' eggs has been mapped to the M1 and M2 genes.See Klimov et al., 1991, Virus Res. 19:105-114. However, these studiesidentified only a single residue in the M1 gene that correlates withincreased viral titer. Accordingly, identification of additional M1residues associated with increased viral titer is needed to permitdesign and construction of recombinant and/or reassortant influenzaviruses with increased replication capacity. These and other unmet needsare provided by the present invention.

4. SUMMARY

The present invention relates to methods and compositions for increasingthe replication capacity of influenza viruses in, for example,embryonated hens' eggs and/or cell culture. The invention is based, inpart, on the identification of particular M1 protein amino acidsassociated with increased replication capacity. By using an M geneencoding an M1 protein that comprises one or more of these particularamino acids, improved influenza viral yields can be achieved.

Accordingly, in a first aspect, the invention provides a method forincreasing the replication capacity of an influenza virus that comprisesaltering an amino acid at a position corresponding to at least one ofposition 95, 143, 144, or 218 of the M1 protein of influenza strainMDV-A, thereby increasing the replication capacity of the influenzavirus. It is specifically contemplated that conservative andnon-conservative amino acid substitutions at these positions are withinthe scope of the invention. In certain embodiments, the amino acidcorresponding to position 218 is not altered to be a threonine (T).

The amino acid(s) of the M1 protein can be altered by any method knownto one skilled in the art, without limitation. In one embodiment, the M1protein is altered by changing the nucleotide sequence of a geneencoding the M1 protein.

In certain embodiments, the amino acid at the position corresponding toposition 95 is altered. In certain embodiments, the amino acid at theposition corresponding to position 95 is altered to be lysine (K). Incertain embodiments, the amino acid at the position corresponding toposition 143 is altered. In certain embodiments, the amino acid at theposition corresponding to position 143 is altered to be alanine (A). Incertain embodiments, the amino acid at the position corresponding toposition 144 is altered. In certain embodiments, the amino acid at theposition corresponding to position 144 is altered to be phenylalanine(F). In certain embodiments, the amino acid at the positioncorresponding to position 218 is altered. In certain embodiments, theamino acid at the position corresponding to position 218 is altered tobe isoleucine (I).

In certain embodiments, the method for increasing the replicationcapacity of an influenza virus comprises altering amino acids at leasttwo positions corresponding to position 95, 143, 144, or 218 of the M1protein of influenza strain MDV-A, thereby increasing the replicationcapacity of the influenza virus. In certain embodiments, the method forincreasing the replication capacity of an influenza virus comprisesaltering amino acids at least three positions corresponding to position95, 143, 144, or 218 of the M1 protein of influenza strain MDV-A,thereby increasing the replication capacity of the influenza virus. Incertain embodiments, the method for increasing the replication capacityof an influenza virus comprises altering amino acids at positionscorresponding to positions 95, 143, 144, and 218 of the M1 protein ofinfluenza strain MDV-A, thereby increasing the replication capacity ofthe influenza virus. In certain embodiments, an amino acid at a positioncorresponding to at least one of position 95, 143, or 144 is altered. Incertain embodiments, an amino acid at a position corresponding to atleast one of position 95, 143, or 144 is altered and an amino acid at aposition corresponding to position 218 is altered. In certainembodiments, the amino acid at the position corresponding to position 95is altered to be lysine (K), the amino acid at the positioncorresponding to position 143 is altered to be alanine (A), the aminoacid at the position corresponding to position 144 is altered to bephenylalanine (F), and the amino acid at the position corresponding toposition 218 is altered to be isoleucine (I).

In certain embodiments, the replication capacity of the influenza virusis increased at least 2-fold relative to the same influenza virus in theabsence of the alteration. In certain embodiments, the replicationcapacity of the influenza virus is increased at least 4-fold relative tothe same influenza virus in the absence of the alteration. In certainembodiments, the replication capacity of the influenza virus isincreased at least 8-fold relative to the same influenza virus in theabsence of the alteration. In certain embodiments, the replicationcapacity of the influenza virus is increased at least 10-fold relativeto the same influenza virus in the absence of the alteration.

In certain embodiments, the influenza virus grows to a titer of at leastabout 7.5 log₁₀ PFU/ml in embryonated eggs and/or cell culture. Incertain embodiments, the influenza virus grows to a titer of at leastabout 8 log₁₀ PFU/ml in embryonated eggs and/or cell culture. In certainembodiments, the influenza virus grows to a titer of at least about 8.5log₁₀ PFU/ml in embryonated eggs and/or cell culture. In certainembodiments, the influenza virus grows to a titer of at least about 9log₁₀ PFU/ml in embryonated eggs and/or cell culture.

The methods of the invention can be advantageously used to producerecombinant and/or reassortant influenza viruses that exhibit increasedreplication capacity relative to parent strains. Such recombinant and/orreassortant viruses can be made, for example, by introducing a pluralityof vectors collectively comprising nucleic acid sequences that expressan infectious influenza viral particle into a population of host cellscapable of supporting viral replication, as extensively described below.The host cells can be cultured under conditions permissive for viralgrowth, and influenza viruses recovered. In certain embodiments, theinfluenza viruses can be attenuated viruses, cold adapted viruses and/ortemperature sensitive viruses. For example, in certain embodiments, therecombinant and/or reassortant influenza viruses can be attenuated, coldadapted, temperature sensitive viruses, such as are suitable foradministration as a live attenuated vaccine, e.g., in a intranasalvaccine formulation.

For example, in some embodiments, the influenza viruses can beartificially engineered influenza viruses comprising one or more aminoacid substitutions which increase influenza virus replication capacity.In certain embodiments, the influenza virus is a recombinant and/orreassortant influenza virus that comprises an M1 protein, wherein anamino acid of the M1 protein at a position corresponding to at least oneof position 95, 143, 144, or 218 of the M1 protein of influenza strainMDV-A is altered. In certain embodiments, an amino acid at a positioncorresponding to at least one of position 95, 143, or 144 is altered. Incertain embodiments, an amino acid at a position corresponding to atleast one of position 95, 143, or 144 is altered and an amino acid at aposition corresponding to position 218 is altered. In certainembodiments, the amino acid at the position corresponding to position 95is altered to be lysine (K). In certain embodiments, the amino acid atthe position corresponding to position 143 is altered to be alanine (A).In certain embodiments, the amino acid at the position corresponding toposition 144 is altered to be phenylalanine (F). In certain embodiments,the amino acid at the position corresponding to position 218 is alteredto be isoleucine (I). In certain embodiments, the amino acids atpositions corresponding to each of positions 95, 143, 144 and 218 of theM1 protein of influenza strain MDV-A are altered. In addition, theinfluenza virus can include one or more additional amino acidalterations not enumerated above.

In certain embodiments, the replication capacity of the recombinantand/or reassortant influenza virus is increased at least 2-fold relativeto the same recombinant and/or reassortant influenza virus in theabsence of the alteration. In certain embodiments, the replicationcapacity of the recombinant and/or reassortant influenza virus isincreased at least 4-fold relative to the same recombinant and/orreassortant influenza virus in the absence of the alteration. In certainembodiments, the replication capacity of the recombinant and/orreassortant influenza virus is increased at least 8-fold relative to thesame recombinant and/or reassortant influenza virus in the absence ofthe alteration. In certain embodiments, the replication capacity of therecombinant and/or reassortant influenza virus is increased at least10-fold relative to the same recombinant and/or reassortant influenzavirus in the absence of the alteration.

In certain embodiments, the recombinant and/or reassortant influenzavirus grows to a titer of at least about 7.5 log₁₀ PFU/ml in embryonatedeggs and/or cell culture. In certain embodiments, the recombinant and/orreassortant influenza virus grows to a titer of at least about 8 log₁₀PFU/ml in embryonated eggs and/or cell culture. In certain embodiments,the recombinant and/or reassortant influenza virus grows to a titer ofat least about 8.5 log₁₀ PFU/ml in embryonated eggs and/or cell culture.In certain embodiments, the recombinant and/or reassortant influenzavirus grows to a titer of at least about 9 log₁₀ PFU/ml in embryonatedeggs and/or cell culture.

In certain embodiments, the recombinant and/or reassortant influenzavirus comprises genomic segments 1, 2, 3, 5, and 8 from influenza strainMDV-A, genomic segment 7 from influenza strain A/Puerto Rico/8/34(A/PR/8/34), and genomic segments 4 and 6 from any influenza strainknown to one skilled in the art without limitation.

The recombinant and/or reassortant influenza viruses can be made by anymethod known to one skilled in the art without limitation. In someembodiments, a plurality of vectors collectively encoding at least the 6internal genome segments (e.g., segments 1-3, 5, 7, and 8; “thebackbone”) of one or more influenza strains along with one or moregenome segments encoding immunogenic influenza surface antigens of adifferent influenza strain can be introduced into a population of hostcells. For example, at least the 6 internal genome segments of aselected influenza strain, for example, a strain including at least oneof the M1 residues associated with increased replication capacity asdescribed above can be introduced into a population of host cells alongwith one or more segments encoding immunogenic surface antigens derivedfrom another virus strain. Typically the immunogenic surface antigensinclude either or both of the hemagglutinin (HA) and/or neuraminidase(NA) antigens. In embodiments where a single segment encoding animmunogenic surface antigen is introduced, the 7 complementary segmentsof the selected virus can be introduced into the host cells. Further,the at least 6 internal genome segments can be selected to have one ormore additional phenotypes as well. For example, the internal genomesegments can be from a selected attenuated, cold adapted and/ortemperature sensitive influenza strain, e.g., a ca, att, ts strain ofA/Ann Arbor/6/60, B/Ann Arbor/1/66, or any other ca, att, and/or tsstrain known to one skilled in the art without limitation.

In certain aspects, the invention provides an expression vectorcomprising a nucleic acid sequence operably linked to a pol I or pol IIpromoter, wherein the nucleic acid sequence encodes an influenza M1protein comprising lysine (K) at position 95, alanine (A) at position143, phenylalanine (F) at position 144, or isoleucine (I) at position218, wherein the positions correspond to the M1 protein of influenzastrain MDV-A. In certain embodiments, the nucleic acid sequence does notencode a wild-type M1 protein of influenza strain A/PR/8/34. In certainembodiments, the M1 protein comprises lysine (K) at position 95, alanine(A) at position 143, phenylalanine (F) at position 144, and isoleucine(I) at position 218.

In some embodiments, the expression vectors of the invention can be bidirectional expression vectors. A bi-directional expression vector ofthe invention typically includes a first promoter and a second promoter,wherein the first and second promoters are operably linked toalternative strands of the same double stranded cDNA encoding the viralnucleic acid comprising a segment of the influenza virus genome, e.g., anucleic acid encoding an M1 protein. Preferably, one of the promoters isa RNA polymerase I promoter and the other promoter is an RNA polymeraseII dependent promoter. The promoters are preferably able to initiatetranscription in the cell into which the vector is to be introduced.Thus, for example, if the vector is to be introduced into a canine cell,a canine RNA pol I and/or RNA pol II promoter can be used. Optionally,the bi-directional expression vector also includes a polyadenylationsignal and/or a terminator sequence. For example, the polyadenylationsignal and/or the terminator sequence can be located flanking a segmentof the influenza virus genome internal to the two promoters. Onefavorable polyadenylation signal in the context of the invention is theSV40 polyadenylation signal. An exemplary plasmid vector of theinvention is the plasmid pAD3000.

Alternatively, the expression vectors of the invention can beunidirectional expression vectors. In a unidirectional vector system ofthe invention, the gene or cDNA encoding an influenza protein, e.g., anM1 protein associated with increased replication capacity as describedherein, is located downstream of a pol I and a pol II promoter. The polII promoter produces capped positive-sense viral mRNA and the pol Ipromoter produces uncapped positive-sense viral cRNA. The promoters arepreferably able to initiate transcription in the cell into which thevector is to be introduced. Thus, for example, if the vector is to beintroduced into a canine cell, a canine RNA pol I and/or RNA pol IIpromoter can be used. Optionally, the unidirectional expression vectoralso includes a polyadenylation signal and/or a terminator sequence. Forexample, the polyadenylation signal and/or the terminator sequence canbe located flanking a segment of the influenza virus genome distal tothe two promoters.

Whether bidirectional, unidirectional, or otherwise, the expressionvectors can be introduced into host cells capable of supporting thereplication of influenza virus from the vector promoters. Favorableexamples of host cells include Vero cells, Per.C6 cells, BHK cells, PCKcells, MDCK cells, MDBK cells, 293 cells (e.g., 293T cells), and COScells. In combination with the pAD3000 plasmid vectors described herein,Vero cells, 293 cells, and COS cells are particularly suitable. In someembodiments, co-cultures of a mixture of at least two of these celllines, e.g., a combination of COS and MDCK cells or a combination of293T and MDCK cells, constitute the population of host cells.

The host cells including the influenza vectors can then be grown inculture under conditions permissive for replication and assembly ofviruses. In embodiments where cold adapted or temperature sensitiveviruses are grown, host cells comprising the expression vectors of theinvention can be cultured at a temperature below about 37° C.,preferably at a temperature equal to, or less than, about 35° C.Typically, the cells are cultured at a temperature between about 32° C.and about 35° C. In some embodiments, the cells are cultured at atemperature between about 32° C. and 34° C., e.g., at about 33° C.Following culture for a suitable period of time to permit replication ofthe virus to high titer, recombinant and/or reassortant cold-adaptedand/or temperature sensitive viruses can be recovered. Optionally, therecovered viruses can be inactivated.

In yet another aspect, the invention also provides broadly applicablemethods of producing recombinant influenza viruses in cell culture byintroducing a plurality of vectors collectively comprising nucleic acidsequences that express an infectious influenza viral particle, whereinone or more vectors encodes an M1 protein comprising one or moreresidues associated with increased replication capacity as describedherein, into a population of host cells capable of supportingreplication of influenza virus, culturing the cells, and recoveringinfluenza viruses.

In certain embodiments, a plurality of expression vectors, e.g.,plasmids, collectively comprising nucleic acid sequences that express aninfectious influenza viral particle can be introduced into a populationof host cells. In certain embodiments, eight expression vectors, each ofwhich comprises a different influenza genomic segment, can be utilizedto introduce a complete influenza genome into the host cells. Theplasmid that encodes the M1 protein generally comprises one or more ofthe residues associated with increased replication capacity as describedherein. Typically, the plasmid vectors of the invention arebi-directional expression vectors, as described above.

In some embodiments, the influenza viruses correspond to an influenza Bvirus. In some embodiments, the influenza viruses correspond to aninfluenza A virus. In certain embodiments, the methods includerecovering recombinant and/or reassortant influenza viruses capable ofeliciting an immune response upon administration, e.g., intranasaladministration, to a subject. In some embodiments, the viruses areinactivated prior to administration. In other embodiments,live-attenuated viruses are administered. Recombinant and reassortantinfluenza A and influenza B viruses produced according to the methods ofthe invention are also contemplated according to the present invention.

In certain embodiments, the viruses made by the methods include anattenuated influenza virus, a cold adapted influenza virus, atemperature sensitive influenza virus, or a virus with any combinationof these desirable properties, in addition to the increased replicationcapacity phenotype as described above. In one embodiment, the influenzavirus comprises one or more proteins from an influenza B/Ann Arbor/1/66strain virus, e.g., a cold adapted, temperature sensitive, attenuatedstrain of B/Ann Arbor/1/66. In another embodiment, the influenza viruscomprises one or more proteins from an influenza A/Ann Arbor/6/60 strainvirus, e.g., a cold adapted, temperature sensitive, attenuated strain ofA/Ann Arbor/6/60. In certain embodiment of the invention, the virusesare artificially engineered influenza viruses comprising one or moresubstituted amino acid which increase the replication capacity of, e.g.,ca A/Ann Arbor/6/60 or ca B/Ann Arbor/1/66. Such substituted amino acidscan include one or more of amino acids corresponding to 95, 143, 144 and218 of the M1 protein of influenza strain MDV-A. Any amino acidsubstitutions at any of these positions resulting in increasedreplication capacity are encompassed by the viruses and methods of theinvention. It will be understood that some influenza A or B viruses mayalready have the recited residues at the indicated positions, e.g., 95K,143A, 144F, and/or 218I. In this case, the substitutions can be usedsuch that the resulting virus will have a substitution at positions 95,143, 144, and 218.

Optionally, reassortant viruses are produced by introducing vectorscollectively encoding the six internal genome segments of a viral strainselected for its favorable properties, in combination with the genomesegments encoding the surface antigens (HA and NA) of a selected, e.g.,pathogenic strain. For example, the HA segment can be selected from apathogenically relevant H1, H3 or B strain, as is routinely performedfor vaccine production. Similarly, the HA segment can be selected froman emerging pathogenic strain such as an H2 strain (e.g., H₂N₂), an H5strain (e.g., H₅N₁) or an H7 strain (e.g., H₇N₇). Alternatively, theseven complementary gene segments of the first strain can be introducedin combination with either the HA or NA encoding segment. In certainembodiments, the internal gene segments are derived from the influenzaB/Ann Arbor/1/66 or the A/Ann Arbor/6/60 strain. In certain embodiments,the internal gene segments, except for the gene segment encoding the M1protein, are derived from the influenza B/Ann Arbor/1/66 or the A/AnnArbor/6/60 strain, while the gene segment encoding the M1 protein isderived from the A/PR/8/34 strain. Alternately, the gene segmentencoding the M1 protein can be derived from the influenza B/AnnArbor/1/66 or the A/Ann Arbor/6/60 strain, wherein the M1 protein hasbeen altered to encode one or more residues associated with increasedreplication capacity, as described herein.

Additionally, the invention provides methods for producing novelinfluenza viruses with desirable properties relevant to vaccineproduction, e.g., influenza viruses that exhibit increased replicationcapacity, as well as influenza vaccines including such novel influenzaviruses. In certain embodiments, a novel influenza A strain virus can beproduced by introducing mutations that result amino acid substitutionsat one or more specified positions demonstrated herein to be importantfor the increased replication capacity phenotype, e.g., positionscorresponding to position 95, 143, 144, or 218 of the M1 protein ofinfluenza strain MDV-A. For example, mutations can be introduced atnucleotide positions resulting in an amino acid substitution at thespecified amino acid position. Any mutation (at one or more of thesepositions) which individually or in combination results in increasedreplication capacity relative to wild type viruses is a suitablemutation in the context of the present invention.

To increase stability of the desired phenotype, a plurality of mutationscan be typically introduced. Following introduction of the selectedmutation(s) into the influenza genome, the mutated influenza genome canbe replicated under conditions in which virus is produced. For example,the mutated influenza virus genome can be replicated in hens' eggs.Alternatively, the influenza virus genome can be replicated in cellculture. In the latter case, the virus can optionally be furtheramplified in hens' eggs to increase the titer. Viruses producedaccording to the methods of the invention are also a feature of theinvention, as are vaccines including such viruses. Similarly, novelrecombinant viral nucleic acids encoding an M1 protein having one ormore mutations at positions corresponding to position 95, 143, 144,and/or 218 of the M1 protein of influenza strain MDV-A, and polypeptideswith such amino acid substitutions are a feature of the invention.

Accordingly, in one aspect, the invention provides an isolated negativesense RNA expressed from an expression vector of the invention. Incertain embodiments, the negative sense RNA encodes an influenza M1protein comprising lysine (K) at position 95, alanine (A) at position143, phenylalanine (F) at position 144, or isoleucine (I) at position218, wherein the positions correspond to the M1 protein of influenzastrain MDV-A. In certain embodiments, the negative sense RNA is not awild-type genomic RNA from influenza strain A/PR/8/34.

In other aspects, the invention provides an influenza M1 proteincomprising lysine (K) at position 95, alanine (A) at position 143,phenylalanine (F) at position 144, or isoleucine (I) at position 218,wherein the positions correspond to the M1 protein of influenza strainMDV-A. In certain embodiments, the protein is not a wild-type M1 proteinof influenza strain A/PR/8/34.

In certain embodiments, the influenza M1 protein comprises lysine (K) atposition 95, alanine (A) at position 143, phenylalanine (F) at position144, and isoleucine (I) at position 218.

Moreover, influenza viruses comprising the mutations of the inventionare a feature of the invention regardless of the method in which theyare produced. That is, the invention encompasses influenza strainsincluding the mutations of the invention, e.g., any influenza virus withan amino acid substitution relative to wild type at one or morepositions selected from positions corresponding to positions 95, 143,144, and 218 of the M1 protein of influenza strain MDV-A. In certainembodiments, the wild-type influenza virus is MDV-A or A/Wyoming/03/03.

Another aspect of the invention relates to novel methods for rescuingrecombinant or reassortant influenza A or influenza B viruses (i.e.,wild type and variant strains of influenza A and/or influenza viruses)having increased replication capacity in hens' eggs and/or cell cultureas described herein from cells in culture. In such embodiments, aplurality of vectors collectively encoding an influenza virus genome,wherein the genome comprises a genomic segment encoding an M proteinassociated with increased replication capacity, can be introduced into apopulation of cells. The cells are grown under conditions permissive forviral replication, e.g., in the case of cold adapted, attenuated,temperature sensitive virus strains, the cells are grown at atemperature below 37° C., preferably at a temperature equal to, or lessthan, 35° C. Typically, the cells are cultured at a temperature between32° C. and 35° C. In some embodiments, the cells are cultured at atemperature between about 32° C. and 34° C., e.g., at about 33° C.Optionally (e.g., for vaccine production), the cells are grown in serumfree medium without any animal-derived products.

In the methods described above, influenza viruses having increasedreplication capacity can be recovered following culture of the hostcells comprising the influenza genome plasmids. In some embodiments, therecovered influenza viruses having increased replication capacity arerecombinant influenza viruses. In some embodiments, the viruses arereassortant influenza viruses having genetic contributions from morethan one parental strain of virus. Optionally, the recovered recombinantor reassortant influenza viruses having increased replication capacitycan be further amplified by passage in cultured cells or in hens' eggs.

Optionally, the recovered influenza viruses having increased replicationcapacity are inactivated. In some embodiments, the recovered influenzaviruses comprise an influenza vaccine. For example, the recoveredinfluenza vaccine can be a reassortant influenza viruses (e.g., 5:3, 6:2or 7:1 reassortant viruses) having increased replication capacity andhaving an HA and/or NA antigen derived from a selected strain ofinfluenza A or influenza B. In certain embodiments, the reassortantviruses have an increased replication capacity phenotype relative to atleast one of the parent strains. In certain embodiments, the reassortantinfluenza viruses have an attenuated phenotype. Optionally, thereassortant viruses are cold adapted and/or temperature sensitive, e.g.,an attenuated, cold adapted or temperature sensitive influenza virus.Such influenza viruses are useful, for example, as live attenuatedvaccines for the prophylactic production of an immune response specificfor a selected, e.g., pathogenic influenza strain. Influenza viruses,e.g., attenuated reassortant viruses, produced according to the methodsof the invention are also contemplated according to the presentinvention.

In another aspect, the invention relates to methods for producing arecombinant influenza virus vaccine involving introducing a plurality ofvectors collectively encoding an influenza virus genome, wherein theencoded M1 protein comprises one or more residues associated withincreased replication capacity as described herein, into a population ofhost cells capable of supporting replication of influenza virus,culturing the host cells, and recovering an influenza virus capable ofeliciting an immune response upon administration to a subject. Theinfluenza vaccines of the invention can comprise either influenza A orinfluenza B strain viruses. In some embodiments, the influenza vaccineviruses include an influenza virus having increased replicationcapacity, an attenuated influenza virus, a cold adapted influenza virus,or a temperature sensitive influenza virus. In certain embodiments, theviruses possess a combination of these desirable properties. In anembodiment, the influenza virus contains an influenza A/Ann Arbor/6/60strain virus with an altered M1 protein resulting in increasedreplication capacity. In another embodiment, the influenza viruscomprises an influenza B/Ann Arbor/1/66 strain virus with an altered M1protein resulting in increased replication capacity. Alternatively, theinfluenza vaccine includes artificially engineered influenza A orinfluenza B viruses comprising at least one substituted amino acidcorresponding to at least one of positions 95, 143, 144, and 218 of theM1 protein of influenza strain MDV-A associated with increasedreplication capacity.

In some embodiments, the virus includes a reassortant influenza virus(e.g., a 5:3, 6:2 or 7:1 reassortant) having viral genome segmentsderived from more than one influenza virus strain. For example, areassortant influenza virus vaccine favorably includes an HA and/or NAsurface antigen derived from a selected strain of influenza A or B, incombination with the internal genome segments of one or more virusstrain(s) selected for its desirable properties with respect to vaccineproduction. Often, it is desirable to select the strain of influenzafrom which the HA and/or NA encoding segments are derived based onpredictions of local or world-wide prevalence of pathogenic strains. Insome cases, the virus strain contributing at least some of the internalgenome segments is an attenuated, cold adapted and/or temperaturesensitive influenza strain, e.g., of A/Ann Arbor/6/60, B/Ann Arbor/1/66,or an artificially engineered influenza strain having one or more aminoacid substitutions resulting in an attenuated, cold adapted and/ortemperature sensitive phenotype. In such embodiments, the straincontributing the segment encoding the M1 protein is advantageouslyselected to be the A/PR/8/34 strain or an artificially engineeredinfluenza strain having one or more amino acid substitutions resultingin an increased replication capacity phenotype, as described herein.

If desired, the influenza viruses, including influenza vaccine viruses,can be inactivated upon recovery.

Influenza virus vaccines, including attenuated live vaccines, producedby the methods of the invention are also contemplated according to thepresent invention. In certain embodiments the influenza virus vaccinesare reassortant virus vaccines.

In yet another aspect, the invention provides kits including one or moreexpression vectors of the invention. Typically, the kits also includeone or more of: a cell line capable of supporting influenza virusreplication, a buffer, a culture medium, an instruction set, a packagingmaterial, and a container. In some embodiments, the kit includes aplurality of expression vectors, each of which includes at least onesegment of an influenza virus genome. For example, the kits can includea plurality of expression vectors each including one of the internalgenome segments of a selected virus strain, e.g., each selected for itsdesirable properties with respect to vaccine production oradministration. For example, the internal genome segments canindependently be selected from a virus strain having anincreased-replication capacity, attenuated, cold adapted and/ortemperature sensitive strain, e.g., A/Ann Arbor/6/60, B/Ann Arbor/1/66,or A/PR/8/34, or an alternative strain with the desired properties, suchas an artificially engineered strain having one or more amino acidsubstitutions as described herein. In one embodiment, the kit includes aexpression vectors comprising members of a library of nucleic acidsencoding variant HA and/or NA antigens.

The present invention also provides cell cultures including at least onecell comprising a plurality of vectors collectively comprising nucleicacid sequences that express an infectious influenza viral particle,wherein one or more of the vectors comprises a genomic segment encodingan M1 protein associated with increased replication capacity asdescribed herein. The culture can also include a cell culture medium. Insome embodiments, the plurality of vectors includes bidirectionalexpression vectors, e.g., comprising a first promoter inserted between asecond promoter and an SV40 polyadenylation site as described above. Forexample, the first promoter and the second promoter can be situated inopposite orientations flanking at least one segment of an influenzavirus. In some embodiments, e.g., when culturing a cold-adapted and/ortemperature sensitive influenza virus, the cell cultures of theinvention can be maintained at a temperature less than or equal to 35°C., such as between about 32° C. and 35° C., typically between about 32°C. and about 34° C., for example, at about 33° C.

In still another aspect, the invention provides a cell culture systemthat comprises a cell culture comprising at least one cell comprising aplurality of vectors collectively encoding a an influenza virus genome,as described above, and a regulator for maintaining the culture at atemperature less than or equal to 35° C. For example, in someembodiments, the regulator maintains the cell culture at a temperaturebetween about 32° C. and 35° C., typically between about 32° C. andabout 34° C., e.g., at about 33° C. Such embodiments are particularlyuseful for culturing, e.g., a temperature-sensitive and/or cold-adaptedinfluenza virus.

In one embodiment, a method is provided for producing influenza virusesin cell culture, the method comprising: i) introducing a plurality ofvectors collectively encoding an influenza virus genome into apopulation of host cells, wherein the influenza virus genome encodes anM1 protein that comprises one or more amino acids associated withincreased replication capacity as described herein, and which populationof host cells is capable of supporting replication of influenza virus;ii) culturing the population of host cells; and, iii) recovering aplurality of influenza viruses. In some embodiments, the a plurality ofvectors comprise one or more genomic segments from an influenza B/AnnArbor/1/66 virus. In some embodiments, the viral genome encodes an M1protein that has lysine (K) at position 95, alanine (A) at position 143,phenylalanine (F) at position 144, or isoleucine (I) at position 218,where the positions correspond to the positions of influenza strainMDV-A.

In one embodiment, a method is provided for producing an influenza viruswith increased replication capacity, the method comprising: (a)introducing into the segment encoding an M gene of an influenza virusgenome at least one mutation at positions corresponding to positions 95,143, 144, and/or 218 of the M1 protein of influenza strain MDV-A; and(b) replicating the mutated influenza virus genome under conditionswhereby virus is produced. In some embodiments, the mutations encodelysine (K) at position 95, alanine (A) at position 143, phenylalanine(F) at position 144, and/or isoleucine (I) at position 218.

In one embodiment the invention provides methods for manipulating theamino acid residues of M1 to increase the ability of an influenza virusto replicate in embryonated hens' eggs and/or cell culture. In someembodiments, the method involves the introduction of amino acid residuessubstitutions in M1 and makes use of methods of producing influenzavirus in cell culture by introducing a plurality of vectors collectivelyencoding an influenza virus genome into a population of host cellscapable of supporting replication of influenza virus, culturing thecells and recovering influenza virus. Preferably, the recoveredinfluenza virus has increased ability to replicate in embryonated hens'eggs and/or cell culture. In another embodiment, the present inventionprovides influenza virus variants with increased ability to replicate inembryonated hens' eggs and/or cell culture when compared to unmodifiedinfluenza viral strains.

5. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 presents the results of influenza plaque assays on MDCK cellsshowing the effects of the M segment on viral replication.

FIG. 2 presents an alignment of the M1 protein from several differentinfluenza viral strains. M1 protein amino acid sequences shown are asfollows: MDVA-M1 (SEQ ID NO:1), PR8-M (SEQ ID NO:2), Sendai-M1 (SEQ IDNO:3), SY97-M1 (SEQ ID NO:4), and WY03-M1 (SEQ ID NO:5).

FIG. 3 presents the results of influenza plaque assays on MDCK cellsshowing the effects of various site-directed mutations introduced intothe M1 protein on replication in hens' eggs.

FIG. 4 presents the results of influenza plaque assays on MDCK cellsshowing the effects of various site-directed mutations introduced intothe M1 protein on replication in hens' eggs.

6. DETAILED DESCRIPTION OF THE INVENTION

One substantial challenge in developing and producing influenza vaccinesis that one or more of the circulating influenza strains may notreplicate well in embryonic hens' eggs. The present invention identifiesseveral amino acid residues which influence the activities of the M1proteins and have identified specific amino acid substitutions which canmodulate these activities. Thus, the present invention provides M1proteins that can increase the replication capacity of influenza in eggsand/or host cells (e.g., Vero or MDCK cells). Specifically the presentinvention discloses amino acid substitutions and combinations of aminoacid substitutions in M1 that can increase viral replication capacity ineggs and/or cell culture. Thus, the present invention provides, forexample, for the use of reverse genetic technology to improve themanufacture of influenza virus vaccines.

In a first aspect, the methods of the invention provide vectors andmethods for increasing the replication capacity of an influenza virusthat comprises altering an amino acid at a position corresponding to atleast one of position 95, 143, 144, or 218 of the M1 protein ofinfluenza strain MDV-A, thereby increasing the replication capacity ofthe influenza virus. The vectors and methods are useful, for example,for making influenza viruses with increased replication capacity ine.g., hens' eggs and/or cell culture.

One skilled in the art will recognize that the exact position of thealtered amino acid or amino acids can vary depending on the particularinfluenza strain used in the vectors, methods, and viruses of theinvention. For example, the M1 protein of a particular influenza strainmay comprise an insertion or deletion in the M gene encoding the M1protein such that the position corresponding to position 95 of the M1protein of MDV-A is found at, for example, residue 93 or 97 of the M1protein of that particular influenza strain. One skilled in the art canreadily recognize whether a particular amino acid position correspondsto a position that, when altered, is associated with increasedreplication capacity using techniques conventional to the art. One suchconventional technique is to align the amino acid sequences of the M1proteins of MDV-A and the particular influenza strain using algorithmsavailable in the art. An example of such an alignment is shown in FIG.2.

Exemplary algorithms that can be used to construct such alignmentsinclude, but are not limited to, the suite of BLAST programs, e.g.,BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, using defaultparameters of an open gap penalty of 11.0, and an extended gap penaltyof 1.0, and utilize the BLOSUM-62 matrix, which is publicly available onthe Internet at the NCBI website, and the CLUSTAL-W program in MacVectorversion 6.5, operated with default parameters, including an open gappenalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM 30similarity matrix. See also Altschul et al., 1990, J. Mol. Biol.215:403-10 (with special reference to the published default setting,i.e., parameters w=4, t=17) and Altschul et al., 1997, Nucleic AcidsRes., 25:3389-3402.

The influenza viruses can be produced, for example, by introducing aplurality of vectors comprising cloned influenza viral genomic segmentsinto host cells, and culturing the cells. When vectors including aninfluenza virus genome are transfected, recombinant viruses suitable asvaccines can be recovered by standard purification procedures. Using thevector system and methods of the invention, reassortant virusesexpressed from the six internal gene segments of a strain selected forits desirable properties with respect to vaccine production and/or viralreplication, and from the immunogenic HA and NA segments from aselected, e.g., pathogenic strain, can be rapidly and efficientlyproduced in tissue culture. Thus, the system and methods describedherein are useful for the rapid production in cell culture ofrecombinant and reassortant influenza A and B viruses, including virusessuitable for use as vaccines, including live attenuated vaccines, suchas vaccines suitable for intranasal administration.

Typically, a single Master Donor Virus (MDV) strain is selected for eachof the A and B subtypes. In the case of a live attenuated vaccine, theMaster Donor Virus strain is typically chosen for its favorableproperties, e.g., temperature sensitivity, cold adaptation, attenuationand/or increased replication capacity, relative to vaccine production.For example, exemplary Master Donor Strains include temperaturesensitive, attenuated, cold adapted, and/or increased replicationcapacity strains of A/Ann Arbor/6/60 and B/Ann Arbor/1/66, respectively.

For example, a selected master donor type A virus (MDV-A), or masterdonor type B virus (MDV-B), can be produced from a plurality of clonedviral cDNAs encoding a viral genome. In an exemplary embodiment,recombinant viruses are produced from eight cloned viral cDNAs. Eightviral cDNAs representing either the selected MDV-A or MDV-B sequences ofPB2, PB1, PA, NP, HA, NA, M and NS are cloned into a bidirectionalexpression vector, such as a plasmid (e.g., pAD3000), such that theviral genomic RNA can be transcribed from an RNA polymerase I (pol I)promoter from one strand and the viral mRNAs can be synthesized from anRNA polymerase II (pol II) promoter from the other strand. Optionally,any gene segment can be modified, including the HA segment (e.g., toremove the multi-basic cleavage site).

Infectious recombinant MDV-A or MDV-B virus is then recovered followingtransfection of plasmids bearing the eight viral cDNAs into appropriatehost cells, e.g., MDCK cells, Vero cells, co-cultured MDCK/293T orMDCK/COS7 cells. Using the plasmids and methods described herein, theinvention is useful, e.g., for generating 6:2 reassortant influenzavaccines by co-transfection of the 6 internal genes (PB1, PB2, PA, NP, Mand NS) of the selected virus (e.g., MDV-A, MDV-B) together with the HAand NA derived from different corresponding type (A or B) influenzaviruses. Alternately, the plasmids and methods described herein can beused, e.g., for generating 5:3 reassortant influenza vaccines byco-transfection of 5 internal genes (PB1, PB2, PA, NP, and NS) of aselected virus (e.g., MDV-A, MDV-B) together with the M gene fromdifferent corresponding type (A or B) influenza viruses and HA and NAderived from the same or different corresponding type (A or B) influenzaviruses as the virus from which the M gene is derived. For example, theHA segment can be selected from a pathogenically relevant H1, H3 or Bstrain, as is routinely performed for vaccine production. Similarly, theHA segment can be selected from a strain with emerging relevance as apathogenic strain such as an H2 strain (e.g., H2N2), an H5 strain (e.g.,H5N1) or an H7 strain (e.g., H7N7). Reassortants expressed from sevengenome segments of the MDV and either the M, HA, or NA gene of aselected strain (7:1 reassortants) can also be produced. In addition,this system is useful for determining the molecular basis of phenotypiccharacteristics, e.g., the attenuated (att), cold adapted (ca),temperature sensitive (ts), and increased replication capacity (irc)phenotypes, relevant to vaccine production.

In another aspect the invention provides methods for manipulating theamino acid residues of M to increase the capacity for an influenza virusto replicate in embryonated hens' eggs and/or cell culture. For example,the methods of the present invention can be use to modulate M proteinactivity to increase the capacity of an influenza virus to replicate ineggs and/or cell culture as described herein. Additionally, theinvention provides influenza viruses with increased capacity toreplicate in embryonated hens' eggs and/or cell culture as describedherein.

6.1 Definitions

Unless defined otherwise, all scientific and technical terms areunderstood to have the same meaning as commonly used in the art to whichthey pertain. For the purpose of the present invention the followingterms are defined below.

The terms “nucleic acid,” “polynucleotide,” “polynucleotide sequence”and “nucleic acid sequence” refer to single-stranded or double-strandeddeoxyribonucleotide or ribonucleotide polymers, or chimeras or analoguesthereof. As used herein, the term optionally includes polymers ofanalogs of naturally occurring nucleotides having the essential natureof natural nucleotides in that they hybridize to single-stranded nucleicacids in a manner similar to naturally occurring nucleotides (e.g.,peptide nucleic acids). Unless otherwise indicated, a particular nucleicacid sequence of this invention encompasses complementary sequences, inaddition to the sequence explicitly indicated.

The term “gene” is used broadly to refer to any nucleic acid associatedwith a biological function. Thus, genes include coding sequences and/orthe regulatory sequences required for their expression. The term “gene”applies to a specific genomic sequence, as well as to a cDNA or an mRNAencoded by that genomic sequence.

Genes also include non-expressed nucleic acid segments that, forexample, form recognition sequences for other proteins. Non-expressedregulatory sequences include “promoters” and “enhancers,” to whichregulatory proteins such as transcription factors bind, resulting intranscription of adjacent or nearby sequences. A “tissue specific”promoter or enhancer is one which regulates transcription in a specifictissue type or cell type, or types.

The term “vector” refers to a nucleic acid, e.g., a plasmid, viralvector, recombinant nucleic acid or cDNA that can be used to introduceheterologous nucleic acid sequences into a cell. The vectors can beautonomously replicating or not autonomously replicating. A vector canalso be a naked RNA polynucleotide, a naked DNA polynucleotide, apolynucleotide composed of both DNA and RNA within the same strand, apoly-lysine-conjugated DNA or RNA, a peptide-conjugated DNA or RNA, aliposome-conjugated DNA, or the like, that are not autonomouslyreplicating. Most commonly, the vectors of the present invention areplasmids.

An “expression vector” is a vector, such as a plasmid, which is capableof promoting expression, as well as replication of a nucleic acidincorporated therein. Typically, the nucleic acid to be expressed is“operably linked” to a promoter and/or enhancer, and is subject totranscription regulatory control by the promoter and/or enhancer.

A “bidirectional expression vector” is typically characterized by twoalternative promoters oriented in the opposite direction relative to anucleic acid situated between the two promoters, e.g., an RNA pol Ipromoter and an RNA pol II promoter, such that expression can beinitiated in both orientations resulting in, e.g., transcription of bothplus (+) or sense strand, and negative (−) or antisense strand RNAs.Alternatively, the bidirectional expression vector can be an ambisensevector, in which the viral mRNA and viral genomic RNA (as a cRNA) areexpressed from the same strand.

A “unidirectional expression vector” is typically characterized by twoalternative promoters oriented in the same direction relative to anucleic acid operatively linked to the two promoters, e.g., an RNA pol Ipromoter and an RNA pol II promoter, such that expression can beinitiated from both promoters resulting in, e.g., transcription of plus(+) strand RNAs. The plus strand RNA expressed from a pol I promoter canhave defined ends suitable for use as, e.g., a viral cRNA. The plusstrand RNA expressed from a pol I promoter can be capped, and,optionally, polyadenylated and is suitable for, e.g., translation ofproteins encoded by the capped RNA.

In the context of the invention, the term “isolated” refers to abiological material, such as a nucleic acid or a protein, which issubstantially free from components that normally accompany or interactwith it in its naturally occurring environment. The isolated materialoptionally comprises material not found with the material in its naturalenvironment, e.g., a cell. For example, if the material is in itsnatural environment, such as a cell, the material has been placed at alocation in the cell (e.g., genome or genetic element) not native to amaterial found in that environment. For example, a naturally occurringnucleic acid (e.g., a coding sequence, a promoter, an enhancer, etc.)becomes isolated if it is introduced by non-naturally occurring means toa locus of the genome (e.g., a vector, such as a plasmid or virusvector, or amplicon) not native to that nucleic acid. Such nucleic acidsare also referred to as “heterologous” nucleic acids.

The term “encode,” in its various grammatical forms as used herein,refers to the ability of a first biological polymer, or collection offirst biological polymers, to contain sequences that can be used to makeother biological polymers having sequences defined by the firstbiological polymer(s). For example, a nucleic acid encodes a polypeptidewhen the nucleic acid comprises a sequence that, when translated,produces a polypeptide having a sequence defined by the nucleic acidsequence according to the conventional genetic code. In another example,a nucleic acid, or collection of nucleic acids, that encodes aninfluenza virus genome, can comprise the complete set of sequencesnecessary to express an infectious influenza particle. In anotherexample, a negative-strand RNA that encodes a polypeptide can be used asa template for synthesis of positive-strand RNA that can be translatedaccording to the genetic code to obtain the polypeptide.

The term “recombinant” indicates that the material (e.g., a nucleic acidor protein) has been artificially or synthetically (non-naturally)altered by human intervention. The alteration can be performed on thematerial within, or removed from, its natural environment or state.Specifically, when referring to a virus, e.g., an influenza virus, thevirus is recombinant when it is produced by the expression of arecombinant nucleic acid.

The term “reassortant,” when referring to a virus, indicates that thevirus includes genetic and/or polypeptide components derived from morethan one parental viral strain or source. For example, a 7:1 reassortantincludes 7 viral genomic segments (or gene segments) derived from afirst parental virus, and a single complementary viral genomic segment,e.g., encoding hemagglutinin or neuraminidase, from a second parentalvirus. A 6:2 reassortant includes 6 genomic segments, most commonly the6 internal genes from a first parental virus, and two complementarysegments, e.g., hemagglutinin and neuraminidase, from a differentparental virus. A 5:3 reassortant includes 5 genomic segments, mostcommonly the PA, PB1, PB2, NP, and NS segments from a first parentalvirus, the M segment from a second parental virus, and the HA and NAsegments from a third parental virus. Generally, at least one of thefirst, second, and third parental viruses can be different from theother two, or the first, second, and third parental viruses can each bedifferent viruses.

The term “introduced” when referring to a heterologous or isolatednucleic acid refers to the incorporation of a nucleic acid into aeukaryotic or prokaryotic cell where the nucleic acid can beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA). The term includes suchmethods as “infection,” “transfection,” “transformation” and“transduction.” In the context of the invention a variety of methods canbe employed to introduce nucleic acids into prokaryotic cells, includingelectroporation, calcium phosphate precipitation, lipid mediatedtransfection (lipofection), etc.

The term “host cell” means a cell which contains a heterologous nucleicacid, such as a vector, and supports the replication and/or expressionof the nucleic acid, and optionally production of one or more encodedproducts including a polypeptide and/or a virus. Host cells can beprokaryotic cells such as E. coli, or eukaryotic cells such as yeast,insect, amphibian, avian or mammalian cells, including human cells.Exemplary host cells in the context of the invention include Vero(African green monkey kidney) cells, Per.C6 cells (human embryonicretinal cells), BHK (baby hamster kidney) cells, primary chick kidney(PCK) cells, Madin-Darby Canine Kidney (MDCK) cells, Madin-Darby BovineKidney (MDBK) cells, 293 cells (e.g., 293T cells), and COS cells (e.g.,COS1, COS7 cells). The term host cell encompasses combinations ormixtures of cells including, e.g., mixed cultures of different celltypes or cell lines (e.g., Vero and CEK cells). A co-cultivation ofelectroporated sf vero cells is described, for example, inPCT/US04/42669 filed Dec. 22, 2004, which is incorporated by referencein its entirety.

The terms “temperature sensitive,” “cold adapted” and “attenuated” arewell known in the art and are used herein according to their ordinarymeanings. For example, the term “temperature sensitive” (“ts”) canindicate that the virus exhibits a 100 fold or greater reduction intiter at 39° C. relative to 33° C. for influenza A strains, and/or thatthe virus exhibits a 100 fold or greater reduction in titer at 37° C.relative to 33° C. for influenza B strains. For example, the term “coldadapted” (“ca”) can indicate that the virus exhibits growth at 25° C.within 100 fold of its growth at 33° C. For example, the term“attenuated” (“att”) can indicate that the virus replicates in the upperairways of ferrets but is not detectable in lung tissues, and/or doesnot cause influenza-like illness in the animal. It will be understoodthat viruses with intermediate phenotypes, i.e., viruses exhibitingtiter reductions less than 100 fold at 39° C. (for A strain viruses) or37° C. (for B strain viruses), exhibiting growth at 25° C. that is morethan 100 fold than its growth at 33° C. (e.g., within 200 fold, 500fold, 1000 fold, 10,000 fold less), and/or exhibit reduced growth in thelungs relative to growth in the upper airways of ferrets (i.e.,partially attenuated) and/or reduced influenza like illness in theanimal, are also useful viruses in some embodiments of the invention.Growth can indicate viral quantity as indicated by titer, plaque size ormorphology, particle density or other measures known to those of skillin the art.

The term “increased replication capacity,” as used herein with referenceto a viral phenotype, means that the virus grows to a greater titer inan appropriate medium, e.g., an embryonated hens' egg or cell culture,relative to a parent virus. For example, a 7:1 or 5:3 reassortant virusthat has an M segment that results in a greater titer of virus grownrelative to an virus with an M segment from one of the parent strainshas an “increased replication capacity” phenotype. In another example, avirus that has been altered in its M gene to encode amino acids thatresult in a greater titer of virus relative to a parent virus that hasnot been altered in its M gene also has an “increased replicationcapacity” phenotype.

The expression “artificially engineered” is used herein to indicate thatthe virus, viral nucleic acid or virally encoded product, e.g., apolypeptide, a vaccine, comprises at least one mutation introduced byrecombinant methods, e.g., site directed mutagenesis, PCR mutagenesis,etc. The expression “artificially engineered” when referring to a virus(or viral component or product) comprising one or more nucleotidemutations and/or amino acid substitutions indicates that the viralgenome or genome segment encoding the virus (or viral component orproduct) is not derived from naturally occurring sources, such as anaturally occurring or previously existing laboratory strain of virusproduced by non-recombinant methods (such as progressive passage at 25°C.), e.g., a wild type or cold adapted A/Ann Arbor/6/60 or B/AnnArbor/1/66 strain.

The term “about,” as used herein, unless otherwise indicated, refers toa value that is no more than 10% above or below the value being modifiedby the term. For example, the term “about 5 μg/kg” means a range of from4.5 μg/kg to 5.5 μg/kg. As another example, “about 1 hour” means a rangeof from 48 minutes to 72 minutes.

The term “encode,” as used herein, refers to the property of a nucleicacid, e.g., deoxyribonucleic acid, to transcribe a complementary nucleicacid, including a nucleic acid that can be translated into apolypeptide. For example, a deoxyribonucleic acid can encode an RNA thatis transcribed from the deoxyribonucleic acid. Similarly, thedeoxyribonucleic acid can encode a polypeptide translated from an RNAtranscribed from the deoxyribonucleic acid.

6.2 Methods for Enhancing Viral Replication Capacity

In one aspect, the present invention provides a method of increasing thecapacity of an influenza virus to replicate in embryonated hens' eggsand/or host cells. Generally, the methods comprise use of an M1 proteincomprising one or more amino acid residues associated with increasedreplication capacity as described herein. The invention further providesinfluenza virus variants with increased ability to replicate inembryonated hens' eggs and/or host cells when compared to influenzavirus with an M protein that does not comprise the one or more aminoacids associated with increased replication capacity. It is specificallycontemplated that the methods of the invention can be utilized toincrease the replication capacity of an influenza virus in a hen eggand/or cell culture and that increased replication capacity variants mayhave increased replication capacity in hens' eggs and/or host cells.Suitable host cells for the replication of influenza virus include,e.g., Vero cells, Per.C6 cells, BHK cells, MDCK cells, 293 cells and COScells, including 293T cells and COS7 cells.

In one embodiment, the method of the invention introduces at least oneamino acid substitution into an M protein which will increase theability of an influenza virus to replicate in eggs and/or host cells byat least about 10%, or by at least about 20%, or by at least about 30%,or by at least about 40%, or by at least about 50%, or by at least about60%, or by at least about 70%, or by at least about 80%, or by at leastabout 90%, or by at least about 100%, or by at least about 200%, or byat least about 300%, or by at least about 400%, or by at least about500% when compared to the unmodified influenza virus. Preferably, themethod of the invention does not significantly alter the antigenicity ofthe substituted influenza virus when compared to the unsubstitutedvirus. In a specific embodiment, the method of the invention reduces theantigenicity of the substituted influenza virus when compared to theunsubstituted virus by less then 10%, or by less then 20%, or by lessthen 30%, or by less then 40%, or by less then 50%, or by less then 60%,or by less then 70%, or by less then 80%, or by less then 90%, or byless then 100%. Methods to determine viral antigenicity are well knownin the art.

In one embodiment, the method of the invention further encompasses anattenuated influenza virus, a cold adapted influenza virus, atemperature sensitive influenza virus, or a virus with any combinationof these desirable properties. Preferably, the viruses encompassed bythe method of the invention include but are not limited to, influenzaB/Ann Arbor/1/66 strain virus, influenza A/Ann Arbor/6/60 strain virus.In another embodiment, the method of the invention introduces vectorsincluding the six internal genes of a viral strain selected for itsfavorable properties regarding vaccine production, e.g., an attenuated,cold adapted, temperature sensitive, and/or increased replicationcapacity strain, in combination with the genome segments encoding thedesired HA and NA surface antigens to produce influenza viruses withincreased ability to replicate in embryonated hens' eggs and/or hostcells and the desired antigenicity provided by the selected HA and NA.In another embodiment, the method of the invention further comprisesmaking and/or using a non-attenuated influenza virus.

In certain embodiments, the method introduces at least one amino acidsubstitution in the M1 protein at positions corresponding to positions95, 143, 144, or 218 of the M1 protein. In one embodiment, amino acidsubstitutions are made at positions 143 and 144 or at positions 95 and218 or at positions 95 and 143, or at positions 95 and 144, or atpositions 143 and 218, or at positions 144 and 218, or at positions 95,143, and 144, or at positions 95, 143, and 218, or at positions 95, 144,and 218, or at positions 143, 144, and 218, or at positions 95, 143, 144and 218. In certain embodiments, the amino acid at the positioncorresponding to position 95 is altered to be lysine (K). In certainembodiments, the amino acid at the position corresponding to position143 is altered to be alanine (A). In certain embodiments, the amino acidat the position corresponding to position 144 is altered to bephenylalanine (F). In certain embodiments, the amino acid at theposition corresponding to position 218 is altered to be isoleucine (I).In another specific embodiment, the changes at these positions can bemade such that a conservative amino acid change is made with respect tothe changes described above.

One skilled in the art would appreciate that in some cases the M1protein will already have the altered amino acid residues at one or moreof the aforementioned positions. In this situation, substitution(s) canbe introduced at any of the remaining non-matching positions.

It is specifically contemplated that conservative amino acidsubstitutions may be made for said amino acid substitutions at positions95, 143, 144 and/or 218 of M1, described supra.

It is well known in the art that “conservative amino acid substitution”refers to amino acid substitutions that substitutefunctionally-equivalent amino acids. For example, one or more aminoacids of a similar polarity can act as functional equivalents and resultin a silent alteration within the amino acid sequence of the peptide.Substitutions that are charge neutral and which replace a residue with asmaller residue may also be considered “conservative substitutions” evenif the residues are in different groups (e.g., replacement ofphenylalanine with the smaller isoleucine). Families of amino acidresidues having similar side chains have been defined in the art.Families of conservative amino acid substitutions include but are notlimited to, non-polar (e.g., Trp, Phe, Met, Leu, Ile, Val, Ala, Pro),uncharged polar (e.g., Gly, Ser, Thr, Asn, Gln, Tyr, Cys),acidic/negatively charged (e.g., Asp, Glu), basic/positively charged(e.g., Arg, Lys, His), beta-branched (e.g., Thr, Val, Ile), residuesthat influence chain orientation (e.g., Gly, Pro) and aromatic (e.g.,Trp, Tyr, Phe, His). The term “conservative amino acid substitution”also refers to the use of amino acid analogs or variants. Guidanceconcerning how to make phenotypically silent amino acid substitutions isprovided in Bowie et al., 1990, Science 247:1306-10.

In one embodiment, a method of increasing the replication capacity of areassortant influenza virus by at least about 10% is provided whereinthe method comprises the steps of: a) comparing the amino acid sequenceof the reassortant influenza virus with the amino acid sequence of adifferent influenza virus that replicates to a higher titer inembryonated eggs and/or cell culture; and b) altering one or more aminoacid of the sequence of the reassortant virus to match the sequence ofthe different influenza virus, thereby producing one or more alteredreassortant viruses, and c) growing the one or more altered reassortantvirus in eggs and/or cell culture. In some embodiments, the amino acidsequence is the sequence of an M protein, e.g., an M1 protein or an M2protein. Influenza viruses produced by these methods are also anembodiment of the invention.

In another specific embodiment, a method of increasing the replicationcapacity of an influenza virus by at least 10% or at least 20% or atleast 50% or at least 80% is provided, the method comprising the stepsof: a) making amino acid substitutions as needed at one or more of thefollowing M1 positions: 95, 143, 144, or 218, such that suchsubstitution, if made, results in a valine residue at position 95, analanine at position 143, a phenylalanine at position 144, and anthreonine at position 218, and b) growing the influenza virus comprisingthe M1 substitutions in eggs and/or cell culture. Immunogeniccompositions and vaccines comprising the influenza viruses of theinvention are also provided. In further embodiments, the method furthercomprises making amino acid substitutions at positions 143 and 144 or atpositions 95 and 218 or at positions 95 and 143, or at positions 95 and144, or at positions 143 and 218, or at positions 144 and 218, or atpositions 95, 143, and 144, or at positions 95, 143, and 218, or atpositions 95, 144, and 218, or at positions 143, 144, and 218, or atpositions 95, 143, 144 and 218.

6.2.1. Methods for Manipulation of Viral Nucleic Acids and Proteins

In the context of the invention, nucleic acids encoding M proteins withsubstitutions associated with increased replication capacity, expressionvectors, influenza virus nucleic acids and/or proteins and the like canbe manipulated according to well known molecular biology techniques.Detailed protocols for numerous such procedures, includingamplification, cloning, mutagenesis, transformation, and the like, aredescribed in, e.g., in Ausubel et al. Current Protocols in MolecularBiology (supplemented through 2000) John Wiley & Sons, New York(“Ausubel”); Sambrook et al. Molecular Cloning—A Laboratory Manual (2ndEd.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,1989 (“Sambrook”), and Berger and Kimmel Guide to Molecular CloningTechniques Methods in Enzymology volume 152 Academic Press, Inc., SanDiego, Calif. (“Berger”).

In addition to the above references, protocols for in vitroamplification techniques, such as the polymerase chain reaction (PCR),the ligase chain reaction (LCR), Qβ-replicase amplification, and otherRNA polymerase mediated techniques (e.g., NASBA), useful e.g., foramplifying cDNA probes of the invention, are found in Mullis et al.(1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide to Methods andApplications (Innis et al. eds) Academic Press Inc. San Diego, Calif.(1990) (“Innis”); Arnheim and Levinson (1990) C&EN 36; The Journal OfNIH Research (1991) 3:81; Kwoh et al. (1989) Proc Natl Acad Sci USA 86,1173; Guatelli et al. (1990) Proc Natl Acad Sci USA 87:1874; Lomell etal. (1989) J Clin Chem 35:1826; Landegren et al. (1988) Science241:1077; Van Brunt (1990) Biotechnology 8:291; Wu and Wallace (1989)Gene 4: 560; Barringer et al. (1990) Gene 89:117, and Sooknanan andMalek (1995) Biotechnology 13:563. Additional methods, useful forcloning nucleic acids in the context of the present invention, includeWallace et al. U.S. Pat. No. 5,426,039. Improved methods of amplifyinglarge nucleic acids by PCR are summarized in Cheng et al. (1994) Nature369:684 and the references therein.

Certain polynucleotides of the invention, e.g., oligonucleotides can besynthesized utilizing various solid-phase strategies includingmononucleotide- and/or trinucleotide-based phosphoramidite couplingchemistry. For example, nucleic acid sequences can be synthesized by thesequential addition of activated monomers and/or trimers to anelongating polynucleotide chain. See e.g., Caruthers, M. H. et al.(1992) Meth Enzymol 211:3.

In lieu of synthesizing the desired sequences, essentially any nucleicacid can be custom ordered from any of a variety of commercial sources,such as The Midland Certified Reagent Company (Midland, Tex.), The GreatAmerican Gene Company (Salt Lake City, Utah), ExpressGen, Inc. (Chicago,Ill.), Operon Technologies, Inc. (Huntsville, Ala.), and many others.

In addition, substitutions of selected amino acid residues in viralpolypeptides can be accomplished by, e.g., site directed mutagenesis.For example, viral polypeptides with amino acid substitutionsfunctionally correlated with desirable phenotypic characteristic, e.g.,an attenuated phenotype, cold adaptation, temperature sensitivity, canbe produced by introducing specific mutations into a viral nucleic acidsegment encoding the polypeptide. Methods for site directed mutagenesisare well known in the art, and described, e.g., in Ausubel, Sambrook,and Berger, supra. Numerous kits for performing site directedmutagenesis are commercially available, e.g., the Chameleon SiteDirected Mutagenesis Kit (Stratagene, La Jolla), and can be usedaccording to the manufacturers instructions to introduce, e.g., one ormore amino acid substitutions, into a genome segment encoding aninfluenza A or B polypeptide, respectively.

6.3 Influenza Viruses with Increased Replication Capacity

In addition, the present invention further provides influenza viruseswith increased replication capacity. Generally, the influenza viruseswith increased replication capacity are recombinant and/or reassortantinfluenza viruses comprising an M1 protein with amino acid substitutionsassociated with increased replication capacity as described herein. Suchviruses can be made, for example, according to the methods describedabove.

Accordingly, in certain embodiments, the invention provides an influenzavirus that comprises an M1 protein, wherein an amino acid of the M1protein at a position corresponding to at least one of position 95, 143,144, or 218 of the M1 protein of influenza strain MDV-A is altered. Incertain embodiments, the amino acid at the position corresponding toposition 95 is altered to be lysine (K). In certain embodiments, theamino acid at the position corresponding to position 143 is altered tobe alanine (A). In certain embodiments, the amino acid at the positioncorresponding to position 144 is altered to be phenylalanine (F). Incertain embodiments, the amino acid at the position corresponding toposition 218 is altered to be isoleucine (I). In certain embodiments,the amino acids at positions corresponding to each of positions 95, 143,144 and 218 of the M1 protein of influenza strain MDV-A are altered. Inaddition, the influenza virus can include one or more additional aminoacid alterations not enumerated above.

In certain embodiments, the replication capacity of the reassortantand/or recombinant influenza virus is increased at least about 10%, orat least about 20%, or at least about 30%, or at least about 40%, or atleast about 50%, or at least about 60%, or at least about 70%, or atleast about 80%, or at least about 90%, or at least about 100%, or atleast about 200%, or at least about 300%, or at least about 400%, or atleast about 500% when compared to the unmodified influenza virus.

In certain embodiments, the replication capacity of the reassortantand/or recombinant influenza virus is increased at least 2-fold relativeto the same reassortant and/or recombinant influenza virus in theabsence of the alteration. In certain embodiments, the replicationcapacity of the reassortant and/or recombinant influenza virus isincreased at least 4-fold relative to the same reassortant and/orrecombinant influenza virus in the absence of the alteration. In certainembodiments, the replication capacity of the reassortant and/orrecombinant influenza virus is increased at least 8-fold relative to thesame reassortant and/or recombinant influenza virus in the absence ofthe alteration. In certain embodiments, the replication capacity of thereassortant and/or recombinant influenza virus is increased at least10-fold relative to the same reassortant and/or recombinant influenzavirus in the absence of the alteration.

In certain embodiments, the reassortant influenza virus grows to a titerof at least about 7.5 log₁₀ PFU/ml in embryonated eggs and/or cellculture. In certain embodiments, the reassortant influenza virus growsto a titer of at least about 8 log₁₀ PFU/ml in embryonated eggs and/orcell culture. In certain embodiments, the reassortant influenza virusgrows to a titer of at least about 8.5 log₁₀ PFU/ml in embryonated eggsand/or cell culture. In certain embodiments, the reassortant influenzavirus grows to a titer of at least about 9 log₁₀ PFU/ml in embryonatedeggs and/or cell culture.

In certain embodiments, the reassortant influenza virus comprisesgenomic segments 1, 2, 3, 5, and 8 from influenza strain MDV-A, genomicsegment 7 from influenza strain A/PR/8/34, and genomic segments 4 and 6from any influenza strain known to one skilled in the art withoutlimitation.

In certain embodiments, the invention provides an influenza viruscomprising at least one amino acid substitution in the M1 protein at aposition corresponding to positions 95, 143, 144, or 218 of the M1protein. In one embodiment, the M1 protein comprises a substitution atpositions 143 and 144, at positions 95 and 218, at positions 95 and 143,at positions 95 and 144, at positions 143 and 218, at positions 144 and218, at positions 95, 143, and 144, at positions 95, 143, and 218, atpositions 95, 144, and 218, at positions 143, 144, and 218, or atpositions 95, 143, 144 and 218. In certain embodiments, the amino acidat the position corresponding to position 95 is altered to be lysine(K). In certain embodiments, the amino acid at the positioncorresponding to position 143 is altered to be alanine (A). In certainembodiments, the amino acid at the position corresponding to position144 is altered to be phenylalanine (F). In certain embodiments, theamino acid at the position corresponding to position 218 is altered tobe isoleucine (I). In another specific embodiment, the changes at thesepositions can be made such that a conservative amino acid change is madewith respect to the changes described above.

One skilled in the art would appreciate that in some cases the M1protein will already have the altered amino acid residues at one or moreof the aforementioned positions. In this situation, the M1 protein cancomprise one or more substitution(s) at any of the remainingnon-matching positions.

It is specifically contemplated that conservative amino acidsubstitutions may be made for said amino acid substitutions at positions95, 143, 144 and/or 218 of M1, described supra.

In one embodiment, the present invention provides modified influenzaviruses which comprise at least one amino acid substitution in M1 whichincreases their replication capacity in embryonated hens' eggs and/orhost cells when compared to the unmodified influenza virus. Preferably,the ability of an influenza variant having increased replicationcapacity to replicate in eggs and/or host cells has been increased by atleast 10%, or by at least 20%, or by at least 30%, or by at least 40%,or by at least 50%, or by at least 60%, or by at least 70%, or by atleast 80%, or by at least 90%, or by at least 100%, or by at least 200%,or by at least 300%, or by at least 400%, or by at least 500% whencompared to the unmodified influenza virus.

In certain embodiment, an influenza variant having increased replicationcapacity further encompasses an attenuated influenza virus, a coldadapted influenza virus, a temperature sensitive influenza virus, or avirus with any combination of these desirable properties. Preferably,the viruses that can be made into influenza variants having increasedreplication capacity include, but are not limited to, influenza B/AnnArbor/1/66 strain viruses or influenza A/Ann Arbor/6/60 strain viruses.It is specifically contemplated that an influenza variants havingincreased replication capacity can be produced by introducing vectorsincluding the six internal genes of a viral strain selected for itsfavorable properties regarding vaccine production, in combination withthe genome segments encoding the desired substituted HA and NA surfaceantigens.

In another specific embodiment, the invention includes reassortantinfluenza viruses comprising a substitution at one or more of thefollowing positions: 95, 143, 144, or 218, wherein the influenza virusgrows to a titer of at least 8.0 log₁₀ PFU/ml, or at least 8.5 log₁₀PFU/ml, or least 9.0 log₁₀ PFU/ml in embryonated eggs and/or cellculture. Immunogenic compositions and vaccines comprising the influenzaviruses of the invention are also provided.

In another specific embodiment, the invention includes reassortantinfluenza viruses comprising a substitution at one or more of thefollowing positions: 95, 143, 144, or 218, wherein the influenza virusgrows to a titer at least 50% higher, or at least 80% higher, or atleast 100% higher than the same reassortant virus not having thesubstitution made.

6.4 Nucleic Acids encoding Influenza M1 Proteins Associated withIncreased Replication Capacity

In another aspect, the invention provides nucleic acids encoding aninfluenza protein that is associated with increased replicationcapacity. The nucleic acids typically encode an M1 protein having aminoacid residues associated with increased replication capacity asdescribed herein.

Accordingly, in certain embodiments, the invention provides an isolatednucleic acid that encodes an M1 protein, wherein an amino acid of the M1protein at a position corresponding to at least one of position 95, 143,144, or 218 of the M1 protein of influenza strain MDV-A is alteredrelative to a parent nucleic acid. In certain embodiments, the aminoacid at the position corresponding to position 95 is altered to belysine (K). In certain embodiments, the amino acid at the positioncorresponding to position 143 is altered to be alanine (A). In certainembodiments, the amino acid at the position corresponding to position144 is altered to be phenylalanine (F). In certain embodiments, theamino acid at the position corresponding to position 218 is altered tobe isoleucine (I). In certain embodiments, nucleic acid encodes an M1protein wherein each of the amino acids at positions corresponding topositions 95, 143, 144 and 218 of the M1 protein of influenza strainMDV-A are altered relative to the parent nucleic acid.

In certain embodiments, the nucleic acid is DNA. In certain embodiments,the nucleic acid is RNA.

In certain embodiments, the nucleic acid encodes an M1 proteincomprising at least one amino acid substitution at a positioncorresponding to positions 95, 143, 144, or 218 of the M1 protein. Inone embodiment, the M1 protein comprises a substitution at positions 143and 144, at positions 95 and 218, at positions 95 and 143, at positions95 and 144, at positions 143 and 218, at positions 144 and 218, atpositions 95, 143, and 144, at positions 95, 143, and 218, at positions95, 144, and 218, at positions 143, 144, and 218, or at positions 95,143, 144 and 218. In certain embodiments, the amino acid at the positioncorresponding to position 95 is altered to be lysine (K). In certainembodiments, the amino acid at the position corresponding to position143 is altered to be alanine (A). In certain embodiments, the amino acidat the position corresponding to position 144 is altered to bephenylalanine (F). In certain embodiments, the amino acid at theposition corresponding to position 218 is altered to be isoleucine (I).In another specific embodiment, the substitution(s) at these positionscan be made such that a conservative amino acid change is made withrespect to the changes described above.

One skilled in the art would appreciate that in some cases the M1protein encoded by the nucleic acid will already have the altered aminoacid residues at one or more of the aforementioned positions. In thissituation, the M1 protein can comprise one or more substitution(s) atany of the remaining non-matching positions.

It is specifically contemplated that conservative amino acidsubstitutions may be made in the nucleic acid encoding any of the aminoacid substitutions at positions corresponding to positions 95, 143, 144and/or 218 of the M1 protein of MDV-A.

6.5 Vectors for Making Influenza Viruses

In another aspect, the invention provides vectors that comprise anucleic acid of the invention, including expression vectors useful forrecombinantly rescuing a virus from cell culture. Generally, theexpression vectors are useful, for example, for rescuing an influenzavirus in cell culture. Typically, at least one of the expression vectorswill encode an influenza M1 protein that comprises one or more aminoacid residues associated with increased replication capacity asdescribed herein.

In accordance with the present invention, in one embodiment, cDNAencoding viral genomic RNA corresponding to each of the eight genomicsegments of influenza (segments may be from different influenza viruses,e.g., 6 from strain X and 2 from strain Y, or 5 from strain X, 2 fromstrain Y, and 1 from strain Z) can be inserted into a recombinant vectorfor manipulation and production of influenza viruses. A variety ofvectors, including viral vectors, plasmids, cosmids, phage, andartificial chromosomes, can be employed in the context of the invention.Typically, for ease of manipulation, the cDNA is inserted into a plasmidvector, providing one or more origins of replication functional inbacterial and eukaryotic cells, and, optionally, a marker convenient forscreening or selecting cells comprising the plasmid sequence. See, e.g.,Neumann et al, 1999, PNAS. USA 96:9345-9350.

In one embodiment, the vectors of the invention are bidirectionalexpression vectors capable of initiating transcription of a viralgenomic segment from the inserted cDNA in either direction, that is,giving rise to both (+) strand and (−) strand viral RNA molecules. Toeffect bi-directional transcription, each of the viral genomic segmentsis inserted into an expression vector having at least two independentpromoters, such that copies of viral genomic RNA are transcribed by afirst RNA polymerase promoter (e.g., an RNA pol I promoter), from onestrand, and viral mRNAs are synthesized from a second RNA polymerasepromoter (e.g., an RNA Pol II promoter). Accordingly, the two promoterscan be arranged in opposite orientations flanking at least one cloningsite (i.e., a restriction enzyme recognition sequence) preferably aunique cloning site, suitable for insertion of viral genomic RNAsegments. Alternatively, an “ambisense” expression vector can beemployed in which the (+) strand mRNA and the (−) strand viral RNA (as acRNA) are transcribed from the same strand of the vector.

To ensure the correct 3′ end of each expressed vRNA or cRNA, each vRNAor cRNA expression vector can comprise a ribozyme sequence orappropriate termination sequence (e.g., human, mouse, primate, or canineRNA polymerase I termination sequence) downstream of the RNA codingsequence. This may be, for example, the hepatitis delta virus genomicribozyme sequence or a functional derivative thereof, or the murine rDNAtermination sequence (Genbank Accession Number M12074). Alternatively,for example, a pol I termination sequence may be employed (Neumann etal., 1994, Virology 202:477-479). The RNA expression vectors may beconstructed in the same manner as the vRNA expression vectors describedin Pleschka et al., 1996, J. Virol. 70:4188-4192; Hoffmann and Webster,2000, J. Gen Virol. 81:2843-2847; Hoffmann et al., 2002, Vaccine20:3165-3170; Fodor et al., 1999, J. Virol. 73:9679-9682; Neumann et al,1999, P.N.A.S. USA 96:9345-9350; and Hoffmann et al., 2000, Virology267:310-317, each of which is hereby incorporated by reference in itsentirety.

In another embodiment, the vectors are unidirectional expressionvectors, wherein viral cDNA is inserted between a pol I promoter and atermination sequences (inner transcription unit). This innertranscription unit is flanked by an RNA polymerase II (pol II) promoterand a polyadenylation site (outer transcription unit). In theunidirectional system, the pol I and pol II promoters are upstream ofthe cDNA and produce positive-sense uncapped cRNA (from the pol Ipromoter) and positive-sense capped mRNA (from the pol II promoter. See,e.g., Hoffmann and Webster, 2000, J. Gen. Virol. 81:2843-2847.

In other systems, viral sequences transcribed by the pol I and pol IIpromoters can be transcribed from different expression vectors. In theseembodiments, vectors encoding each of the viral genomic segments underthe control of a pol I promoter (“vRNA expression vectors”) and vectorsencoding one or more viral polypeptides, e.g., influenza PA, PB1, PB2,and NP polypeptides (“protein expression vectors”) under the control ofa pol II promoter can be used.

In either case, with regard to the pol II promoter, the influenza virusgenome segment to be expressed can be operably linked to an appropriatetranscription control sequence (promoter) to direct mRNA synthesis. Avariety of promoters are suitable for use in expression vectors forregulating transcription of influenza virus genome segments. In certainembodiments, the cytomegalovirus (CMV) DNA dependent RNA Polymerase II(Pol II) promoter is utilized. If desired, e.g., for regulatingconditional expression, other promoters can be substituted which induceRNA transcription under the specified conditions, or in the specifiedtissues or cells. Numerous viral and mammalian, e.g., human promotersare available, or can be isolated according to the specific applicationcontemplated. For example, alternative promoters obtained from thegenomes of animal and human viruses include such promoters as theadenovirus (such as Adenovirus 2), papilloma virus, hepatitis-B virus,and polyoma virus, and various retroviral promoters. Mammalian promotersinclude, among many others, the actin promoter, immunoglobulinpromoters, heat-shock promoters, and the like. In a specific embodiment,the regulatory sequence comprises the adenovirus 2 major late promoterlinked to the spliced tripartite leader sequence of human adenovirus 2,as described by Berg et al, Bio Techniques 14:972-978. In addition,bacteriophage promoters can be employed in conjunction with the cognateRNA polymerase, e.g., the T7 promoter. Expression vectors used toexpress viral proteins, in particular viral proteins for RNP complexformation, will preferably express viral proteins homologous to thedesired virus. The expression of viral proteins by these expressionvectors may be regulated by any regulatory sequence known to those ofskill in the art. The regulatory sequence may be a constitutivepromoter, an inducible promoter or a tissue-specific promoter. Furtherexamples of promoters which may be used to control the expression ofviral proteins in protein expression vectors include, but are notlimited to, the SV40 early promoter region (Bernoist and Chambon, 1981,Nature 290:304-310), the promoter contained in the 3′ long terminalrepeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797),the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl.Acad. Sci. USA 78:1441-1445), the regulatory sequences of themetallothionein gene (Brinster et al., 1982, Nature 296:39-42);prokaryotic expression vectors such as the β-lactamase promoter(VIIIa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731),or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA80:21-25); see also “Useful proteins from recombinant bacteria” inScientific American, 1980, 242:74-94; plant expression vectorscomprising the nopaline synthetase promoter region (Herrera-Estrella etal., Nature 303:209-213) or the cauliflower mosaic virus 35S RNApromoter (Gardner et al, 1981, Nucl. Acids Res. 9:2871), and thepromoter of the photosynthetic enzyme ribulose biphosphate carboxylase(Herrera-Estrella et al., 1984, Nature 310:115-120); promoter elementsfrom yeast or other fungi such as the Gal 4 promoter, the ADC (alcoholdehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkalinephosphatase promoter, and the following animal transcriptional controlregions, which exhibit tissue specificity and have been utilized intransgenic animals: elastase I gene control region which is active inpancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Omitz etal., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald,1987, Hepatology 7:425-515); insulin gene control region which is activein pancreatic beta cells (Hanahan, 1985, Nature 315:115-122),immunoglobulin gene control region which is active in lymphoid cells(Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444),mouse mammary tumor virus control region which is active in testicular,breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495),albumin gene control region which is active in liver (Pinkert et al.,1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control regionwhich is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol.5:1639-1648; Hammer et al., 1987, Science 235:53-58; alpha 1-antitrypsingene control region which is active in the liver (Kelsey et al., 1987,Genes and Devel. 1: 161-171), beta-globin gene control region which isactive in myeloid cells (Mogram et al., 1985, Nature 315:338-340;Kollias et al., 1986, Cell 46:89-94; myelin basic protein gene controlregion which is active in oligodendrocyte cells in the brain (Readheadet al., 1987, Cell 48:703-712), myosin light chain-2 gene control regionwhich is active in skeletal muscle (Sani, 1985, Nature 314:283-286), andgonadotropic releasing hormone gene control region which is active inthe hypothalamus (Mason et al., 1986, Science 234: 1372-1378).

In a specific embodiment, protein expression vectors of the inventioncomprise a promoter operably linked to a nucleic acid sequence, one ormore origins of replication, and, optionally, one or more selectablemarkers (e.g., an antibiotic resistance gene). In another embodiment, aprotein expression vector of the invention that is capable of producingbicistronic mRNA may be produced by inserting bicistronic mRNA sequence.Certain internal ribosome entry site (IRES) sequences may be utilized.Preferred IRES elements include, but are not limited to the mammalianBiP IRES and the hepatitis C virus IRES.

In one embodiment, a nucleic acid of the invention is inserted intoplasmid pAD3000 or a derivative thereof. See, U.S. patent applicationpublication US2005/0266026, hereby incorporated by reference in itsentirety. Thus, in certain embodiments, the expression vector is abi-directional expression vector. In certain embodiments, the expressionvector comprises a SV40 polyadenylation signal flanking a segment of theinfluenza virus genome internal to the two promoters. In certainembodiments, the expression vector comprises the cytomegalovirus (CMV)DNA dependent RNA Pol II promoter.

Vectors containing gene inserts can be identified by, e.g., threegeneral approaches: (a) nucleic acid hybridization; (b) presence orabsence of “marker” gene functions; and, in the case of expressionvectors, (c) expression of inserted sequences. In the first approach,the presence of the viral gene inserted in an vector(s) can be detectedby nucleic acid hybridization using probes comprising sequences that arehomologous to the inserted gene(s). In the second approach, therecombinant vector/host system can be identified and selected based uponthe presence or absence of certain “marker” gene functions (e.g.,resistance to antibiotics or transformation phenotype) caused by theinsertion of the gene(s) in the vector(s). In the third approach,expression vectors can be identified by assaying the gene productexpressed. Such assays can be based, for example, on the physical orfunctional properties of the viral protein in in vitro assay systems,e.g., binding of viral proteins to antibodies.

In a specific embodiment, one or more protein expression vectors encodeand express the viral proteins necessary for the formation of RNPcomplexes. In another embodiment, one or more protein expression vectorsencode and express the viral proteins necessary to form viral particles.In yet another embodiment, one or more protein expression vectors encodeand express the all of the viral proteins of a particularnegative-strand RNA virus.

Transcription from expression vectors can optionally be increased byincluding an enhancer sequence. Enhancers are typically short, e.g.,10-500 bp, cis-acting DNA elements that act in concert with a promoterto increase transcription. Many enhancer sequences have been isolatedfrom mammalian genes (hemoglobin, elastase, albumin, alpha.-fetoprotein,and insulin), and eukaryotic cell viruses. The enhancer can be splicedinto the vector at a position 5′ or 3′ to the heterologous codingsequence, but is typically inserted at a site 5′ to the promoter.Typically, the promoter, and if desired, additional transcriptionenhancing sequences are chosen to optimize expression in the host celltype into which the heterologous DNA is to be introduced (Scharf et al.(1994) Heat stress promoters and transcription factors Results ProblCell Differ 20:125-62; Kriegler et al. (1990) Assembly of enhancers,promoters, and splice signals to control expression of transferred genesMethods in Enzymol 185: 512-27). Optionally, the amplicon can alsocontain a ribosome binding site or an internal ribosome entry site(IRES) for translation initiation.

The expression vectors of the invention can also include sequences forthe termination of transcription and for stabilizing the mRNA, such as apolyadenylation site or a termination sequence (e.g., human, mouse,primate, or canine RNA polymerase I termination sequence). Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. In someembodiments, the SV40 polyadenylation sequences provide apolyadenylation signal.

In addition, as described above, the vectors optionally include one ormore selectable marker genes to provide a phenotypic trait for selectionof transformed host cells, in addition to genes previously listed,markers such as dihydrofolate reductase or neomycin resistance aresuitable for selection in eukaryotic cell culture.

The expression vector containing the appropriate DNA sequence asdescribed above, as well as an appropriate promoter or control sequence,can be employed to transform a host cell permitting expression of theprotein. While the expression vectors of the invention can be replicatedin bacterial cells, most frequently it will be desirable to introducethem into mammalian cells, e.g., Vero cells, BHK cells, MDCK cell, 293cells, or COS cells, or co-cultures thereof, for the purpose ofexpression.

The expression vectors of the invention can be used to directing theexpressing of genomic vRNA(s) or corresponding cRNA(s) which have one ormore mutations (e.g., removal or inactivation of a polybasic cleavagesite in the HA gene of particular influenza pandemic strains such asH₅N₁). These mutations may result in the attenuation of the virus. Forexample, the vRNA segments may be the vRNA segments of an influenza Avirus having an attenuated base pair substitution in a pan-handle duplexpromoter region, in particular, for example, the known attenuating basepair substitution of A for C and U for G at position 11-12′ in theduplex region of the NA-specific vRNA (Fodor et al., 1998, J. Virol.6923-6290).

Most commonly, the genome segment encoding the influenza virus proteinfurther includes any additional sequences necessary for its expression,including translation into a functional viral protein. In othersituations, a minigene, or other artificial construct encoding the viralproteins, e.g., an HA or NA or M1 protein, can be employed. In thiscase, it is often desirable to include specific initiation signals whichaid in the efficient translation of the heterologous coding sequence.These signals can include, e.g., the ATG initiation codon and adjacentsequences. To insure translation of the entire insert, the initiationcodon is inserted in the correct reading frame relative to the viralprotein. Exogenous transcriptional elements and initiation codons can beof various origins, both natural and synthetic. The efficiency ofexpression can be enhanced by the inclusion of enhancers appropriate tothe cell system in use.

If desired, polynucleotide sequences encoding additional expressedelements, such as signal sequences, secretion or localization sequences,and the like can be incorporated into the vector, usually in-frame withthe polynucleotide sequence of interest, e.g., to target polypeptideexpression to a desired cellular compartment, membrane, or organelle, orinto the cell culture media. Such sequences are known to those of skillin the art, and include secretion leader peptides, organelle targetingsequences (e.g., nuclear localization sequences, ER retention signals,mitochondrial transit sequences), membrane localization/anchor sequences(e.g., stop transfer sequences, GPI anchor sequences), and the like.

Finally, any of the vectors described in U.S. Pat. Nos. 6,951,754,6,887,699, 6,649,372, 6,544,785, 6,001,634, 5,854,037, 5,824,536,5,840,520, 5,820,871, 5,786,199, and 5,166,057 and U.S. PatentApplication Publication Nos. 20060019350, 20050158342, 20050037487,20050266026, 20050186563, 20050221489, 20050032043, 20040142003,20030035814, and 20020164770, for example, can be used in accordancewith the present invention. Generally, the vectors described in thesepublications can be adapted for use in accordance with the presentinvention by introducing a nucleic acid of the invention (e.g., anucleic acid encoding an influenza M1 protein with substitutionsassociated with increased replication capacity as described herein) intoexpression vectors to direct synthesis of viral vRNA or cRNA.

The expression vectors can also be used to make chimeric viruses havingincreased replication capacity that express sequences heterologous to aviral genome. In such embodiments, expression vectors directing theexpression of vRNA(s) or corresponding cRNA(s) can be introduced intohost cells along with expression vectors directing the expression ofviral proteins, including an M1 protein comprising residues associatedwith increased replication capacity, to generate novel infectiousrecombinant negative-strand RNA viruses or chimeric viruses. See, e.g.,US patent application publication no. US2004/0002061. Heterologoussequences which may be engineered into these viruses include, forexample, antisense nucleic acids and a nucleic acid such as a ribozyme.Alternatively, heterologous sequences which express a peptide orpolypeptide may be engineered into these viruses. Heterologous sequencesencoding the following peptides or polypeptides may be engineered intothese viruses include: 1) antigens that are characteristic of apathogen; 2) antigens that are characteristic of autoimmune disease; 3)antigens that are characteristic of an allergen; and 4) antigens thatare characteristic of a tumor. For example, heterologous gene sequencesthat can be engineered into the chimeric viruses of the inventioninclude, but are not limited to, epitopes of human immunodeficiencyvirus (HIV) such as gp 160; hepatitis B virus surface antigen (HBsAg);the glycoproteins of herpes virus (e.g., gD, gE); VP1 of poliovirus; andantigenic determinants of nonviral pathogens such as bacteria andparasites to name but a few.

Antigens that are characteristic of autoimmune disease typically will bederived from the cell surface, cytoplasm, nucleus, mitochondria and thelike of mammalian tissues, including antigens characteristic of diabetesmellitus, multiple sclerosis, systemic lupus erythematosus, rheumatoidarthritis, pernicious anemia, Addison's disease, scleroderma, autoimmuneatrophic gastritis, juvenile diabetes, and discoid lupus erythromatosus.

Antigens that are allergens are generally proteins or glycoproteins,including antigens derived from pollens, dust, molds, spores, dander,insects and foods.

Antigens that are characteristic of tumor antigens typically will bederived from the cell surface, cytoplasm, nucleus, organelles and thelike of cells of tumor tissue. Examples include antigens characteristicof tumor proteins, including proteins encoded by mutated oncogenes;viral proteins associated with tumors; and glycoproteins. Tumorsinclude, but are not limited to, those derived from the types of cancer:lip, nasopharynx, pharynx and oral cavity, esophagus, stomach, colon,rectum, liver, gall bladder, pancreas, larynx, lung and bronchus,melanoma of skin, breast, cervix, uterine, ovary, bladder, kidney,uterus, brain and other parts of the nervous system, thyroid, prostate,testes, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma andleukemia.

In one specific embodiment of the invention, the heterologous sequencesare derived from the genome of human immunodeficiency virus (HIV),preferably human immunodeficiency virus-1 or human immunodeficiencyvirus-2. In another embodiment of the invention, the heterologous codingsequences may be inserted within an negative-strand RNA virus genecoding sequence such that a chimeric gene product is expressed whichcontains the heterologous peptide sequence within the viral protein. Insuch an embodiment of the invention, the heterologous sequences may alsobe derived from the genome of a human immunodeficiency virus, preferablyof human immunodeficiency virus-1 or human immunodeficiency virus-2.

In instances whereby the heterologous sequences are HIV-derived, suchsequences may include, but are not limited to sequences derived from theenv gene (i.e., sequences encoding all or part of gp160, gp120, and/orgp41), the pol gene (i.e., sequences encoding all or part of reversetranscriptase, endonuclease, protease, and/or integrase), the gag gene(i.e., sequences encoding all or part of p7, p6, p55, p17/18, p24/25)tat, rev, nef, vif, vpu, vpr, and/or vpx.

One approach for constructing these hybrid molecules is to insert theheterologous coding sequence into a DNA complement of a negative-strandRNA virus gene so that the heterologous sequence is flanked by the viralsequences required for viral polymerase activity; e.g., an RNA pol Ipromoter and a polyadenylation site. In an alternative approach,oligonucleotides encoding an RNA pol I promoter, e.g., the complement ofthe 3′-terminus or both termini of the virus genomic segments can beligated to the heterologous coding sequence to construct the hybridmolecule. The placement of a foreign gene or segment of a foreign genewithin a target sequence was formerly dictated by the presence ofappropriate restriction enzyme sites within the target sequence.However, advances in molecular biology have lessened this problemgreatly. Restriction enzyme sites can readily be placed anywhere withina target sequence through the use of site-directed mutagenesis (e.g.,see, for example, the techniques described by Kunkel, 1985, Proc. Natl.Acad. Sci. U.S.A. 82:488). Variations in polymerase chain reaction (PCR)technology, described, also allow for the specific insertion ofsequences (i.e., restriction enzyme sites) and allow for the facileconstruction of hybrid molecules. Alternatively, PCR reactions could beused to prepare recombinant templates without the need of cloning. Forexample, PCR reactions could be used to prepare double-stranded DNAmolecules containing a DNA-directed RNA polymerase promoter (e.g.,bacteriophase T3, T7 or SP6) and the hybrid sequence containing theheterologous gene and a canine RNA pol I promoter. RNA templates couldthen be transcribed directly from this recombinant DNA. In yet anotherembodiment, the recombinant vRNAs or corresponding cRNAs may be preparedby ligating RNAs specifying the negative polarity of the heterologousgene and the canine RNA pol I promoter using an RNA ligase.

Bicistronic mRNA could be constructed to permit internal initiation oftranslation of viral sequences and allow for the expression of foreignprotein coding sequences from the regular terminal initiation site.Alternatively, a bicistronic mRNA sequence may be constructed whereinthe viral sequence is translated from the regular terminal open readingframe, while the foreign sequence is initiated from an internal site.Certain internal ribosome entry site (IRES) sequences may be utilized.The IRES sequences which are chosen should be short enough to notinterfere with virus packaging limitations. Thus, it is preferable thatthe IRES chosen for such a bicistronic approach be no more than 500nucleotides in length, with less than 250 nucleotides being preferred.Further, it is preferable that the IRES utilized not share sequence orstructural homology with picornaviral elements. Preferred IRES elementsinclude, but are not limited to the mammalian BiP FRES and the hepatitisC virus IRES.

Alternatively, a foreign protein may be expressed from an internaltranscriptional unit in which the transcriptional unit has an initiationsite and polyadenylation site. In another embodiment, the foreign geneis inserted into a negative-strand RNA virus gene such that theresulting expressed protein is a fusion protein.

6.6 Viral Propagation in Cell Culture

The present invention further contemplates that influenza viruses havinginfluenza viruses having increased replication capacity may be grown incell culture, as extensively described below.

6.6.1. Cells and Cell Cultures for Growing Influenza Viruses

Any host cell known to one skilled in the art to be useful for culturinginfluenza viruses may be used in accordance with the present invention.Host cells are preferably animal cells, more preferably mammalian cells,and most preferably canine cells. Host cells which may be used togenerate the negative-strand RNA viruses of the invention includeprimary cells, cultured or secondary cells, and transformed orimmortalized cells (e.g., 293 cells, 293T cells, CHO cells, Vero cells,PK, MDBK, OMK and MDCK cells). In some embodiments, influenza viruseshaving increased replication capacity are generated in MDCK cells.

Typically, propagation of the virus can be accomplished in the mediacompositions in which the host cell is commonly cultured. Suitable hostcells for the replication of influenza virus include, e.g., Vero cells,Per.C6 cells, BHK cells, MDCK cells, 293 cells and COS cells, including293T cells and COS7 cells. Co-cultures including two of the above celllines, e.g., MDCK cells and either 293T or COS cells can also beemployed at a ratio, e.g., of 1:1, to improve replication efficiency.Typically, cells are cultured in a standard commercial culture medium,such as Dulbecco's modified Eagle's medium supplemented with serum(e.g., 10% fetal bovine serum), or in serum free medium, undercontrolled humidity and CO₂ concentration suitable for maintainingneutral buffered pH (e.g., at pH between 7.0 and 7.2). Suitable serumfree media are described, for example, in U.S. Provisional ApplicationNo. 60/638,166, filed Dec. 23, 2004, and in U.S. Provisional ApplicationNo. 60/641,139, filed Jan. 5, 2005, each of which is hereby incorporatedby reference in its entirety. Optionally, the medium containsantibiotics to prevent bacterial growth, e.g., penicillin, streptomycin,etc., and/or additional nutrients, such as L-glutamine, sodium pyruvate,non-essential amino acids, additional supplements to promote favorablegrowth characteristics, e.g., trypsin, β-mercaptoethanol, and the like.

Procedures for maintaining mammalian cells in culture have beenextensively reported, and are known to those of skill in the art.General protocols are provided, e.g., in Freshney (1983) Culture ofAnimal Cells: Manual of Basic Technique, Alan R. Liss, New York; Paul(1975) Cell and Tissue Culture, 5^(th) ed., Livingston, Edinburgh; Adams(1980) Laboratory Techniques in Biochemistry and Molecular Biology-CellCulture for Biochemists, Work and Burdon (eds.) Elsevier, Amsterdam.Additional details regarding tissue culture procedures of particularinterest in the production of influenza virus in vitro include, e.g.,Merten et al. (1996) Production of influenza virus in cell cultures forvaccine preparation. In Cohen and Shafferman (eds) Novel Strategies inDesign and Production of Vaccines, which is incorporated herein in itsentirety. Additionally, variations in such procedures adapted to thepresent invention are readily determined through routineexperimentation.

Cells for production of influenza virus can be cultured inserum-containing or serum free medium. In some case, e.g., for thepreparation of purified viruses, it is desirable to grow the host cellsin serum free conditions. Cells can be cultured in small scale, e.g.,less than 25 ml medium, culture tubes or flasks or in large flasks withagitation, in rotator bottles, or on microcarrier beads (e.g.,DEAE-Dextran microcarrier beads, such as Dormacell, Pfeifer & Langen;Superbead, Flow Laboratories; styrene copolymer-tri-methylamine beads,such as Hillex, SoloHill, Ann Arbor) in flasks, bottles or reactorcultures. Microcarrier beads are small spheres (in the range of 100-200microns in diameter) that provide a large surface area for adherent cellgrowth per volume of cell culture. For example a single liter of mediumcan include more than 20 million microcarrier beads providing greaterthan 8000 square centimeters of growth surface. For commercialproduction of viruses, e.g., for vaccine production, it is oftendesirable to culture the cells in a bioreactor or fermenter. Bioreactorsare available in volumes from under 1 liter to in excess of 100 liters,e.g., Cyto3 Bioreactor (Osmonics, Minnetonka, Minn.); NBS bioreactors(New Brunswick Scientific, Edison, N.J.); laboratory and commercialscale bioreactors from B. Braun Biotech International (B. Braun Biotech,Melsungen, Germany).

6.6.2. Methods for Growing Influenza Viruses in Cell Culture

The present invention further provides methods of generating infectiousrecombinant influenza viruses having increased replication capacity byintroducing protein expression vectors and vRNA or corresponding cRNAexpressing expression vectors of the invention into host cells in theabsence of helper virus. The present invention also provides methods ofgenerating infectious recombinant influenza viruses having increasedreplication capacity by introducing protein expression vectors and vRNAor corresponding cRNA expressing expression vectors of the inventioninto host cells in the presence of helper virus. In either case,recombinant influenza viruses having increased replication capacity canbe generated using vectors encoding an M1 protein with residuesassociated with increased replication capacity as described herein.

Protein expression vectors and expression vectors directing theexpression of vRNAs or corresponding cRNAs can be introduced into hostcells using any technique known to those of skill in the art withoutlimitation. For example, expression vectors of the invention can beintroduced into host cells by employing electroporation, DEAE-dextran,calcium phosphate precipitation, liposomes, microinjection, andmicroparticle-bombardment (see, e.g., Sambrook et al., MolecularCloning: A Laboratory Manual, 2 ed., 1989, Cold Spring Harbor Press,Cold Spring Harbor, N.Y.). The expression vectors may be introduced intohost cells simultaneously or sequentially.

In one embodiment, one or more expression vectors directing theexpression of vRNA(s) or corresponding cRNA(s) are introduced into hostcells prior to the introduction of expression vectors directing theexpression of viral proteins. In another embodiment, one or moreexpression vectors directing the expression of viral proteins areintroduced into host cells prior to the introduction of the one or moreexpression vectors directing the expression of vRNA(s) or correspondingcRNA(s). In accordance with these embodiments, the expression vectorsdirecting the expression of the vRNA(s) or corresponding cRNA(s) mayintroduced together or separately in different transfections. Further,in accordance with these embodiments, the expression vectors directingthe expression of the viral proteins can be introduced together orseparately in different transfections.

In another embodiment, one or more expression vectors directing theexpression of vRNA(s) or corresponding cRNA(s) and one or moreexpression vectors directing the expression of viral proteins areintroduced into host cells simultaneously. In certain embodiments, allof the expression vectors are introduced into host cells usingliposomes.

Appropriate amounts and ratios of the expression vectors for carryingout a method of the invention may be determined by routineexperimentation. As guidance, in the case of liposomal transfection orcalcium precipitation of plasmids into the host cells, it is envisagedthat each plasmid may be employed at a few μgs, e.g., 1 to 10 μg, forexample, diluted to a final total DNA concentration of about 0.1 μg/mlprior to mixing with transfection reagent in conventional manner. It maybe preferred to use vectors expressing NP and/or RNA-dependent RNApolymerase subunits at a higher concentration than those expressing vRNAsegments. One skilled in the art will appreciate that the amounts andratios of the expression vectors may vary depending upon the host cells.

In one embodiment, at least 0.5 μg, preferably at least 1 μg, at least2.5 μg, at least 5 μg, at least 8 μg, at least 10 μg, at least 15 μg, atleast 20 μg, at least 25 μg, or at least 50 μg of one or more proteinexpression vectors of the invention are introduced into host cells togenerate infectious recombinant negative-strand RNA virus. In anotherembodiment, at least 0.5 μg, preferably at least 1 μg, at least 2.5 μg,at least 5 μg, at least 8 μg, at least 10 μg, at least 15 μg, at least20 μg, at least 25 μg or at least 50 μg of one or more expressionvectors of the invention directing the expression of vRNAs or cRNAs areintroduced into host cells to generate infectious influenza viruses.

The present invention further provides methods of generating infectiousrecombinant influenza viruses having increased replication capacity instably transduced host cell lines. The stably transduced host cell linesof the invention may be produced by introducing cDNA encoding, interalia, an influenza M1 protein having residues associated with increasedreplication capacity, controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcription terminationsequences, polyadenylation sites, etc.), and a selectable marker intohost cells. Following the introduction of the foreign DNA, thetransduced cells may be allowed to grow for 1-2 days in an enrichedmedia, and then are switched to a selective media. The selectable markerconfers resistance to the cells and allows the cells to stably integratethe DNA into their chromosomes. Transduced host cells with the DNAstably integrated can be cloned and expanded into cell lines.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler et al., 1980,Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981, Proc. Natl. Acad.Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid(Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, whichconfers resistance to the aminoglycoside G-418 (Colberre-Garapin et al.,1981, J. Mol. Biol. 150:1); and hygro, which confers resistance tohygromycin (Santerre et al., 1984, Gene 30:147) genes.

The infectious recombinant influenza viruses generated by methods of theinvention which are not attenuated, may be attenuated or killed by, forexample, classic methods. For example, recombinant influenza viruses ofthe invention may be killed by heat or formalin treatment, so that thevirus is not capable of replicating. Recombinant influenza viruses ofthe invention which are not attenuated may be attenuated by, e.g.,passage through unnatural hosts to produce progeny viruses which areimmunogenic, but not pathogenic.

Attenuated, live or killed viruses produced in accordance with theinvention may subsequently be formulated into a vaccine composition inconventional manner or used to produce additional virus, e.g., in eggs.Where such a virus has a chimeric vRNA segment as discussed above whichencodes a foreign antigen, it may be formulated to achieve vaccinationagainst more than one pathogen simultaneously. Attenuated recombinantviruses produced in accordance with the invention which possess achimeric vRNA segment may also be designed for other therapeutic uses,e.g., an anti-tumor agent or gene therapy tool, in which case productionof the virus will be followed by its incorporation into an appropriatepharmaceutical composition together with a pharmaceutically acceptablecarrier or diluent.

Helper virus free rescue in accordance with the invention isparticularly favored for generation of reassortant influenza viruses,especially reassortant influenza viruses desired for vaccine useparticularly since selection methods are not needed to rid the cultureof helper virus.

The methods of the present invention may be modified to incorporateaspects of methods known to those skilled in the art, in order toimprove efficiency of rescue of infectious viral particles. For example,the reverse genetics technique involves the preparation of syntheticrecombinant viral RNAs that contain the non-coding regions of thenegative strand virus RNA which are essential for the recognition byviral polymerases and for packaging signals necessary to generate amature virion. The recombinant RNAs are synthesized from a recombinantDNA template and reconstituted in vitro with purified viral polymerasecomplex to form recombinant ribonucleoprotein (RNPs) which can be usedto transfect cells. A more efficient transfection is achieved if theviral polymerase proteins are present during transcription of thesynthetic RNAs either in vitro or in vivo. The synthetic recombinantRNPs can be rescued into infectious virus particles. The foregoingtechniques are described in U.S. Pat. No. 5,166,057 issued Nov. 24,1992; in U.S. Pat. No. 5,854,037 issued Dec. 29, 1998; in U.S. Pat. No.5,789,229 issued Aug. 4, 1998; in European Patent Publication EP0702085A1, published Feb. 20, 1996; in U.S. patent application Ser. No.09/152,845; in International Patent Publications PCR WO97/12032published Apr. 3, 1997; WO96/34625 published Nov. 7, 1996; in WO99/02657published Jan. 21, 1999; WO98/53078 published Nov. 26, 1998; WO98/02530published Jan. 22, 1998; WO99/15672 published Apr. 1, 1999; WO98/13501published Apr. 2, 1998; WO97/06720 published Feb. 20, 1997; and EPO 780475 published Jun. 25, 1997, each of which is incorporated by referenceherein in its entirety.

The present invention further provides a method for generating incultured cells an infectious recombinant influenza virus, such as aninfluenza A or B virus, said method comprising: (a) introducing into apopulation of cells capable of supporting growth of said virus a firstset of expression vectors capable of expressing in said cells genomicvRNA segments to provide the complete genomic vRNA segments of saidvirus, wherein the genomic vRNA segment encoding the M1 protein encodesan M1 protein having one or more residues associated with increasedreplication capacity as described herein; (b) introducing into saidcells a second set of expression vectors capable of expressing mRNAencoding one or more polypeptides of said virus; and (c) culturing saidcells whereby infectious influenza viral particles are produced. Incertain embodiments, the recombinant virus is influenza A or B virus. Incertain embodiments, the first set of expression vectors comprises 1-8plasmids. In certain embodiments, the first set of expression vectorscomprises one plasmid. In certain embodiments, the second set ofexpression vectors comprises 1-8 plasmids. In certain embodiments, thesecond set of expression vectors comprises one plasmid. In certainembodiments, the first, second, or both sets of expression vectors areintroduced by electroporation. In certain embodiments, the first set ofexpression vectors encode each vRNA segment of an influenza virus. Incertain embodiments, the second set of expression vectors encode themRNA of one or more or all influenza polypeptides. In certainembodiments, the first set or second set of expression vectors (or bothsets) encode a vRNA or mRNA of a second virus. For instance, a set ofvectors can comprise one or more vectors encoding the HA and/or NA mRNAand/or vRNA of a second influenza virus. In one embodiment, helper virusis used in the method.

The present invention further provides a method for generating incultured cells infectious recombinant influenza viruses, such as aninfluenza A or B virus, said method comprising: (a) introducing into apopulation of cells capable of supporting growth of said virus a set ofexpression vectors capable of both expressing in said cells genomic vRNAsegments to provide the complete genomic vRNA segments of said virus andof expressing mRNA encoding one or more polypeptides of said virus,wherein the genomic vRNA segment encoding the M1 protein encodes an M1protein having one or more residues associated with increasedreplication capacity as described herein; and (b) culturing said cellswhereby viral particles are produced. In certain embodiments, theinfluenza virus is influenza A or B virus. In certain embodiments, theset of expression vectors comprises 1-17 plasmids. In certainembodiments, the set of expression vectors comprises 1-8 plasmids. Incertain embodiments, the set of expression vectors comprises 1-3plasmids. In certain embodiments, the set of expression vectorscomprises one plasmid. In certain embodiments, the sets of expressionvectors are introduced by electroporation. In certain embodiments, theset of expression vectors encode each vRNA segment of an influenzavirus. In certain embodiments, the set of expression vectors encode themRNA of one or more influenza polypeptides. In certain embodiments, theset of expression vectors encode each vRNA segment of an influenza virusand the mRNA of one or more influenza polypeptides. In certainembodiments, the set of expression vectors comprise a nucleic acid ofthe invention, for example, a nucleic acid encoding an influenza M1protein having one or more residues associated with increasedreplication capacity as described herein. In certain embodiments, theset of expression vectors encode a vRNA or mRNA of a second virus. Forinstance, in some embodiments, the set of vectors can comprise one ormore vectors encoding the HA and/or NA mRNA and/or vRNA of a secondinfluenza virus. In certain embodiments, the first set or second set ofexpression vectors (or both sets) encode a vRNA or mRNA of a secondvirus. For instance, in some embodiments, a set of vectors can compriseone or more vectors encoding the HA and/or NA mRNA and/or vRNA of asecond influenza virus. In one embodiment, helper virus is used in themethod. In other embodiments, a set of vectors can comprise one or morevectors encoding the M1 of a second influenza virus, e.g., an M1 proteinassociated with increased replication capacity as described herein or anM1 protein from a A/PR/8/34 influenza virus.

The present invention further provides a method for generating incultured cells infectious recombinant influenza viral particles, saidmethod comprising: (a) introducing into a population of cells capable ofsupporting growth of said virus a first set of expression vectorscapable of expressing in said cells genomic vRNA to provide the completegenomic vRNA of said virus, wherein the genomic vRNA segment encodingthe M1 protein encodes an M1 protein having one or more residuesassociated with increased replication capacity as described herein; (b)introducing into said cells a second set of expression vectors capableof expressing mRNA encoding one or more polypeptides of said virus; and(c) culturing said cells whereby said viral particles are produced. Incertain embodiments, the cells are canine cells. In certain embodiments,the cells are MDCK cells. In certain embodiments, the virus is influenzaA or B virus. In certain embodiments, the first set of expressionvectors comprises 1-8 plasmids. In certain embodiments, the first set ofexpression vectors comprises one plasmid. In certain embodiments, thesecond set of expression vectors comprises 1-8 plasmids. In certainembodiments, the second set of expression vectors comprises one plasmid.In certain embodiments, the first, second, or both sets of expressionvectors are introduced by electroporation. In certain embodiments, thefirst set of expression vectors encode each vRNA segment of an influenzavirus. In certain embodiments, the second set of expression vectorsencode the mRNA of one or more influenza polypeptides. In certainembodiments, the first set or second set of expression vectors (or bothsets) comprise a nucleic acid of the invention, for example, a nucleicacid encoding an influenza M1 protein having one or more residuesassociated with increased replication capacity as described herein. Inone embodiment, helper virus is used in the method. In one embodiment,the cultured cells used in the method are canine cells.

The present invention further provides a method for generating incultured cells infectious viral particles of an influenza virus, saidmethod comprising: (a) introducing into a population of cells capable ofsupporting growth of said virus a set of expression vectors capable ofboth expressing in said cells genomic vRNA to provide the completegenomic vRNA of said virus and expressing mRNA encoding one or morepolypeptides of said virus, wherein the genomic vRNA segment encodingthe M1 protein encodes an M1 protein having one or more residuesassociated with increased replication capacity as described herein; and(b) culturing said cells whereby said viral particles are produced. Incertain embodiments, the cells are canine cells. In certain embodiments,the cells are MDCK cells. In certain embodiments, the virus is aminfluenza A or B virus. In certain embodiments, the set of expressionvectors comprises 1-17 plasmids. In certain embodiments, the set ofexpression vectors comprises 1-8 plasmids. In certain embodiments, theset of expression vectors comprises 1-3 plasmids. In certainembodiments, the set of expression vectors is introduced byelectroporation. In certain embodiments, the set of expression vectorsencode each vRNA segment of an influenza virus. In certain embodiments,the set of expression vectors encode the mRNA of one or more influenzapolypeptides. In certain embodiments, the set of expression vectorsencode each vRNA segment of an influenza virus and the mRNA of one ormore influenza polypeptides. In certain embodiments, the set ofexpression vectors comprise a nucleic acid of the invention, forexample, a nucleic acid encoding an influenza M1 protein having one ormore residues associated with increased replication capacity asdescribed herein. In certain embodiments, the set of expression vectorsencode a vRNA or mRNA of a second virus. For instance, in someembodiments, the set of vectors can comprises one or more vectorsencoding the HA and/or NA and/or M1 mRNA and/or vRNA of a secondinfluenza virus. In one embodiment, helper virus is used in the method.In one embodiment, the cultured cells used in the method are caninecells.

In a specific embodiment, the present invention provides for methods ofgenerating infectious recombinant influenza virus in host cells usingexpression vectors to express the vRNA segments or corresponding cRNAsand influenza virus proteins, in particular PB1, PB2, PA and NA. Inaccordance with this embodiment, helper virus may or may not be includedto generate the infectious recombinant influenza viruses.

The infectious recombinant influenza viruses of the invention may or maynot replicate and produce progeny. Preferably, the infectiousrecombinant influenza viruses of the invention are attenuated.Attenuated infectious recombinant influenza viruses may, for example,have a mutation in the NS1 gene.

In certain embodiments, an infectious recombinant influenza virus of theinvention can be used to produce other viruses useful to prepare avaccine composition of the invention. In one embodiment, recombinant orreassortant influenza viruses produced by a method of the invention areused for the production of additional virus for use as a vaccine. Forexample, a population of recombinant or reassortant viruses havingincreased replication capacity can be produced by the methods of theinvention as described above. Subsequently, the population of virusescan be grown in eggs or another culture such that additional viruses areproduced for the preparation of vaccines or an immunogenic composition.

In certain embodiments, the infectious recombinant influenza viruses ofthe invention express heterologous (i.e., non-influenza virus)sequences, as described above. In another embodiment, the infectiousrecombinant influenza viruses of the invention express influenza virusproteins from different influenza strains. In yet another embodiment,the infectious recombinant influenza viruses of the invention expressfusion proteins.

6.6.3. Methods for Recovering Influenza Viruses from Cell Culture

Viruses can typically be recovered from the culture medium, in whichinfected (transfected) cells have been grown. Typically crude medium isclarified prior to concentration of influenza viruses. Common methodsinclude filtration, ultrafiltration, adsorption on barium sulfate andelution, and centrifugation. For example, crude medium from infectedcultures can first be clarified by centrifugation at, e.g., 1000-2000×gfor a time sufficient to remove cell debris and other large particulatematter, e.g., between 10 and 30 minutes. Alternatively, the medium isfiltered through a 0.8 μm cellulose acetate filter to remove intactcells and other large particulate matter. Optionally, the clarifiedmedium supernatant is then centrifuged to pellet the influenza viruses,e.g., at 15,000×g, for approximately 3-5 hours. Following resuspensionof the virus pellet in an appropriate buffer, such as STE (0.01 MTris-HCl; 0.15 M NaCl; 0.0001 M EDTA) or phosphate buffered saline (PBS)at pH 7.4, the virus is concentrated by density gradient centrifugationon sucrose (60%-12%) or potassium tartrate (50%-10%). Either continuousor step gradients, e.g., a sucrose gradient between 12% and 60% in four12% steps, are suitable. The gradients are centrifuged at a speed, andfor a time, sufficient for the viruses to concentrate into a visibleband for recovery. Alternatively, and for most large scale commercialapplications, virus is elutriated from density gradients using azonal-centrifuge rotor operating in continuous mode. Additional detailssufficient to guide one of skill through the preparation of influenzaviruses from tissue culture are provided, e.g., in Furminger. VaccineProduction, in Nicholson et al. (eds) Textbook of Influenza pp. 324-332;Merten et al. (1996) Production of influenza virus in cell cultures forvaccine preparation, in Cohen & Shafferman (eds) Novel Strategies inDesign and Production of Vaccines pp. 141-151, and U.S. Pat. No.5,690,937, U.S. publication application nos. 20040265987, 20050266026and 20050158342, which are incorporated by reference herein. If desired,the recovered viruses can be stored at −80° C. in the presence ofsucrose-phosphate-glutamate (SPG) as a stabilizer.

6.7 Influenza Vaccines

Historically, influenza virus vaccines have been produced in embryonatedhens' eggs using strains of virus selected based on empiricalpredictions of relevant strains. More recently, reassortant viruses havebeen produced that comprise selected hemagglutinin and neuraminidaseantigens in the context of an approved attenuated, temperature sensitivemaster strain. Following culture of the virus through multiple passagesin hens' eggs, influenza viruses are recovered and, optionally,inactivated, e.g., using formaldehyde and/or β-propiolactone. However,production of influenza vaccine in this manner has several significantdrawbacks. Among these drawbacks are the inability of certaincirculating influenza strains to replicate to high titer in hens' eggs,thus limiting the amount of vaccine that can be made from the eggs.

The present invention provides a vector system, compositions, andmethods for producing recombinant and reassortant viruses that haveincreased replication capacity in eggs and/or culture which make itpossible to produce vaccines corresponding to one or many selectedantigenic strains of virus. In particular, conditions and strains areprovided that result in efficient production of viruses from a multiplasmid system in cell culture. Optionally, if desired, the viruses canbe further amplified in hens' eggs or cell cultures that differ from thecultures used to rescue the virus.

Reassortant influenza viruses can be readily obtained by introducing asubset of vectors comprising cDNA that encodes genomic segments of amaster influenza virus, in combination with complementary segmentsderived from strains of interest (e.g., antigenic variants of interest).Typically, the master strains are selected on the basis of desirableproperties relevant to vaccine administration. For example, for vaccineproduction, e.g., for production of a live attenuated vaccine, themaster donor virus strain may be selected for an attenuated phenotype,cold adaptation and/or temperature sensitivity. In this context,influenza A strain ca A/Ann Arbor/6/60; influenza B strain ca B/AnnArbor/1/66; or another strain selected for its desirable phenotypicproperties, e.g., an attenuated, cold adapted, and/or temperaturesensitive strain, are favorably selected as master donor strains.

In one embodiment, plasmids comprising cDNA encoding the six internalvRNA segments of the influenza master virus strain, (i.e., PB1, PB2, PA,NP, NB, M1, BM2, NS1 and NS2) are transfected into suitable host cellsin combination with cDNA encoding hemagglutinin and neuraminidase vRNAsegments from an antigenically desirable strain, e.g., a strainpredicted to cause significant local or global influenza infection.Following replication of the reassortant virus in cell, reassortantviruses are recovered. Optionally, if desired, the viruses can befurther amplified in hens' eggs or cell cultures that differ from thecultures used to rescue the virus. Optionally, the recovered virus canbe inactivated using a denaturing agent such as formaldehyde or13-propiolactone.

6.7.1. Vaccine Compositions

The present invention further provides compositions comprising aninfluenza virus of the invention. Such compositions can comprise, forexample, recombinant and reassortant viruses in an appropriate carrieror excipient. Typically, the carrier or excipient is a pharmaceuticallyacceptable carrier or excipient, such as sterile water, aqueous salinesolution, aqueous buffered saline solutions, aqueous dextrose solutions,aqueous glycerol solutions, ethanol, allantoic fluid from uninfectedhens' eggs (i.e., normal allantoic fluid “NAF”) or combinations thereof.The preparation of such solutions insuring sterility, pH, isotonicity,and stability is effected according to protocols established in the art.Generally, a carrier or excipient is selected to minimize allergic andother undesirable effects, and to suit the particular route ofadministration, e.g., subcutaneous, intramuscular, intranasal, etc.

6.7.2. Methods of Administering Vaccine Compositions

Generally, the influenza viruses of the invention are administered in aquantity sufficient to stimulate an immune response specific for one ormore strains of influenza virus. Preferably, administration of theinfluenza viruses elicits a protective immune response. Dosages andmethods for eliciting a protective immune response against one or moreinfluenza strains are known to those of skill in the art. For example,inactivated influenza viruses are provided in the range of about 1-1000HID₅₀ (human infectious dose), i.e., about 10⁵-10⁸ pfu (plaque formingunits) per dose administered. Alternatively, about 10-50 μg, e.g., about15 μg HA can be administered without an adjuvant, with smaller dosesbeing administered with an adjuvant. Typically, the dose can be adjustedwithin this range based on, e.g., age, physical condition, body weight,sex, diet, time of administration, and other clinical factors. Theprophylactic vaccine formulation can be systemically administered, e.g.,by subcutaneous or intramuscular injection using a needle and syringe,or a needleless injection device. Alternatively, the vaccine formulationcan be administered intranasally, either by drops, large particleaerosol (greater than about 10 microns), or spray into the upperrespiratory tract. While any of the above routes of delivery results ina protective systemic immune response, intranasal administration confersthe added benefit of eliciting mucosal immunity at the site of entry ofthe influenza virus. For intranasal administration, attenuated livevirus vaccines are often preferred, e.g., an attenuated, cold adaptedand/or temperature sensitive recombinant or reassortant influenza virus.While stimulation of a protective immune response with a single dose ispreferred, additional dosages can be administered, by the same ordifferent route, to achieve the desired prophylactic effect.

Alternatively, an immune response can be stimulated by ex vivo or invivo targeting of dendritic cells with influenza viruses. For example,proliferating dendritic cells are exposed to viruses in a sufficientamount and for a sufficient period of time to permit capture of theinfluenza antigens by the dendritic cells. The cells are thentransferred into a subject to be vaccinated by standard intravenoustransplantation methods.

Optionally, the formulation for prophylactic administration of theinfluenza viruses, or subunits thereof, also contains one or moreadjuvants for enhancing the immune response to the influenza antigens.Suitable adjuvants include: saponin, mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil or hydrocarbon emulsions, bacilleCalmette-Guerin (BCG), Corynebacterium parvum, and the syntheticadjuvants QS-21 and MF59.

If desired, prophylactic vaccine administration of influenza viruses canbe performed in conjunction with administration of one or moreimmunostimulatory molecules. Immunostimulatory molecules include variouscytokines, lymphokines and chemokines with immunostimulatory,immunopotentiating, and pro-inflammatory activities, such asinterleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-12, IL-13); growthfactors (e.g., granulocyte-macrophage (GM)-colony stimulating factor(CSF)); and other immunostimulatory molecules, such as macrophageinflammatory factor, Flt3 ligand, B7.1, B7.2, etc. The immunostimulatorymolecules can be administered in the same formulation as the influenzaviruses, or can be administered separately. Either the protein or anexpression vector encoding the protein can be administered to produce animmunostimulatory effect.

In another embodiment, the vectors of the invention including influenzagenome segments can be employed to introduce heterologous nucleic acidsinto a host organism or host cell, such as a mammalian cell, e.g., cellsderived from a human subject, in combination with a suitablepharmaceutical carrier or excipient as described above. Typically, theheterologous nucleic acid is inserted into a non-essential region of agene or gene segment, e.g., the M gene of segment 7. The heterologouspolynucleotide sequence can encode a polypeptide or peptide, or an RNAsuch as an antisense RNA or ribozyme. The heterologous nucleic acid canthen be introduced into a host or host cells by producing recombinantviruses comprising the heterologous nucleic acid, and the viruses areadministered as described above. In one embodiment, the heterologouspolynucleotide sequence is not derived from an influenza virus.

Alternatively, a vector of the invention including a heterologousnucleic acid can be introduced and expressed in a host cells byco-transfecting the vector into a cell infected with an influenza virus.Optionally, the cells are then returned or delivered to the subject,typically to the site from which they were obtained. In someapplications, the cells are grafted onto a tissue, organ, or system site(as described above) of interest, using established cell transfer orgrafting procedures. For example, stem cells of the hematopoieticlineage, such as bone marrow, cord blood, or peripheral blood derivedhematopoietic stem cells can be delivered to a subject using standarddelivery or transfusion techniques.

Alternatively, the viruses comprising a heterologous nucleic acid can bedelivered to the cells of a subject in vivo. Typically, such methodsinvolve the administration of vector particles to a target cellpopulation (e.g., blood cells, skin cells, liver cells, neural(including brain) cells, kidney cells, uterine cells, muscle cells,intestinal cells, cervical cells, vaginal cells, prostate cells, etc.,as well as tumor cells derived from a variety of cells, tissues and/ororgans. Administration can be either systemic, e.g., by intravenousadministration of viral particles, or by delivering the viral particlesdirectly to a site or sites of interest by a variety of methods,including injection (e.g., using a needle or syringe), needlelessvaccine delivery, topical administration, or pushing into a tissue,organ or skin site. For example, the viral vector particles can bedelivered by inhalation, orally, intravenously, subcutaneously,subdermally, intradermally, intramuscularly, intraperitoneally,intrathecally, by vaginal or rectal administration, or by placing theviral particles within a cavity or other site of the body, e.g., duringsurgery.

The above described methods are useful for therapeutically and/orprophylactically treating a disease or disorder by introducing a vectorof the invention comprising a heterologous polynucleotide encoding atherapeutically or prophylactically effective polypeptide (or peptide)or RNA (e.g., an antisense RNA or ribozyme) into a population of targetcells in vitro, ex vivo or in vivo. Typically, the polynucleotideencoding the polypeptide (or peptide), or RNA, of interest is operablylinked to appropriate regulatory sequences as described above in thesections entitled “Expression Vectors.” Optionally, more than oneheterologous coding sequence is introduced into a single vector orvirus. For example, in addition to a polynucleotide encoding atherapeutically or prophylactically active polypeptide or RNA, thevector can also include additional therapeutic or prophylacticpolypeptides, e.g., antigens, co-stimulatory molecules, cytokines,antibodies, etc., and/or markers, and the like.

In one embodiment, the invention provides compositions comprisingreassortant and recombinant viruses of the invention (or portionsthereof) that have been treated with an agent such as benzonase, toeliminate potential oncogenes. Accordingly, an oncogene-free vaccinecomposition is specifically included within the embodiments of theinvention.

The methods and vectors of the present invention can be used totherapeutically or prophylactically treat a wide variety of disorders,including genetic and acquired disorders, e.g., as vaccines forinfectious diseases, due to viruses, bacteria, and the like.

6.8 Specific Embodiments

Additional embodiments of the present invention are presented in Table1.

TABLE 1 Specific embodiments 1 A method for increasing the replicationcapacity of an influenza virus, comprising altering an amino acid at aposition corresponding to at least one of position 95, 143, 144 or 218of the M1 protein of influenza strain MDV-A, thereby increasing thereplication capacity of the influenza virus, with the proviso that theamino acid corresponding to position 218 is not altered to be athreonine (T). 2 The method of embodiment 1, wherein an amino acidcorresponding to at least two of positions 95, 143, 144, or 218 arealtered. 3 The method of embodiment 1, wherein an amino acidcorresponding to at least three of positions 95, 143, 144, or 218 arealtered. 4 The method of embodiment 1, wherein the amino acid at theposition corresponding to position 95 is altered. 5 The method ofembodiment 4, wherein the amino acid at the position corresponding toposition 95 is altered to be lysine (K). 6 The method of embodiment 1,wherein the amino acid at the position corresponding to position 143 isaltered. 7 The method of embodiment 6, wherein the amino acid at theposition corresponding to position 143 is altered to be alanine (A). 8The method of embodiment 1, wherein the amino acid at the positioncorresponding to position 144 is altered. 9 The method of embodiment 8,wherein the amino acid at the position corresponding to position 144 isaltered to be phenylalanine (F). 10 The method of embodiment 1, whereinthe amino acid at the position corresponding to position 218 is altered.11 The method of embodiment 10, wherein the amino acid at the positioncorresponding to position 218 is altered to be isoleucine (I). 12 Amethod for increasing the replication capacity of an influenza virus,comprising altering amino acids at positions corresponding to positions95, 143, 144 and 218 of the M1 protein of influenza strain MDV-A,thereby increasing the replication capacity of the influenza virus. 13The method of embodiment 12, wherein the amino acid at the positioncorresponding to position 95 is altered to be lysine (K). 14 The methodof embodiment 4, wherein the amino acid at the position corresponding toposition 143 is altered to be alanine (A). 15 The method of embodiment4, wherein the amino acid at the position corresponding to position 144is altered to be phenylalanine (F). 16 The method of embodiment 4,wherein the amino acid at the position corresponding to position 218 isaltered to be isoleucine (I). 17 The method of embodiment 4, wherein theamino acid at the position corresponding to position 95 is altered to belysine (K), the amino acid at the position corresponding to position 143is altered to be alanine (A), the amino acid at the positioncorresponding to position 144 is altered to be phenylalanine (F) and theamino acid at the position corresponding to position 218 is altered tobe isoleucine (I). 18 The method of embodiment 1 or 12, wherein thereplication capacity of the influenza virus is increased at least 2-foldrelative to the same influenza virus in the absence of the alteration.19 The method of embodiment 1 or 12, wherein the replication capacity ofthe influenza virus is increased at least 4-fold relative to the sameinfluenza virus in the absence of the alteration. 20 The method ofembodiment 1 or 12, wherein the replication capacity of the influenzavirus is increased at least 8-fold relative to the same influenza virusin the absence of the alteration. 21 The method of embodiment 1 or 12,wherein the replication capacity of the influenza virus is increased atleast 10-fold relative to the same influenza virus in the absence of thealteration. 22 The method of embodiment 1 or 12, wherein the influenzavirus grows to a titer of at least about 7.5 log10 PFU/ml in embryonatedeggs. 23 The method of embodiment 1 or 12, wherein the influenza virusgrows to a titer of at least about 8 log10 PFU/ml in embryonated eggs.24 The method of embodiment 1 or 12, wherein the influenza virus growsto a titer of at least about 8.5 log10 PFU/ml in embryonated eggs. 25The method of embodiment 1 or 12, wherein the influenza virus grows to atiter of at least about 9 log10 PFU/ml in embryonated eggs. 26 Areassortant influenza virus comprising an M1 protein, wherein an aminoacid of the M1 protein at a position corresponding to at least one ofposition 95, 143, 144, or 218 of the M1 protein of influenza strainMDV-A is altered. 27 The reassortant influenza virus of embodiment 26,wherein the amino acid at the position corresponding to position 95 isaltered to be lysine (K). 28 The reassortant influenza virus ofembodiment 26, wherein the amino acid at the position corresponding toposition 143 is altered to be alanine (A). 29 The reassortant influenzavirus of embodiment 26, wherein the amino acid at the positioncorresponding to position 144 is altered to be phenylalanine (F). 30 Thereassortant influenza virus of embodiment 26, wherein the amino acid atthe position corresponding to position 218 is altered to be isoleucine(I). 31 The reassortant influenza virus of embodiment 26, wherein theamino acids at positions corresponding to each of positions 95, 143,144, and 218 of the M1 protein of influenza strain MDV-A are altered. 32The reassortant influenza virus of embodiment 26, wherein thereplication capacity of the reassortant influenza virus is increased atleast 2-fold relative to the same reassortant influenza virus in theabsence of the alteration. 33 The reassortant influenza virus ofembodiment 26, wherein the replication capacity of the reassortantinfluenza virus is increased at least 4-fold relative to the samereassortant influenza virus in the absence of the alteration. 34 Thereassortant influenza virus of embodiment 26, wherein the replicationcapacity of the reassortant influenza virus is increased at least 8-foldrelative to the same reassortant influenza virus in the absence of thealteration. 35 The reassortant influenza virus of embodiment 26, whereinthe replication capacity of the reassortant influenza virus is increasedat least 10-fold relative to the same reassortant influenza virus in theabsence of the alteration. 36 The reassortant influenza virus ofembodiment 26, wherein the reassortant influenza virus grows to a titerof at least about 7.5 log10 PFU/ml in embryonated eggs. 37 Thereassortant influenza virus of embodiment 26, wherein the reassortantinfluenza virus grows to a titer of at least about 8 log10 PFU/ml inembryonated eggs. 38 The reassortant influenza virus of embodiment 26,wherein the reassortant influenza virus grows to a titer of at leastabout 8.5 log10 PFU/ml in embryonated eggs. 39 The reassortant influenzavirus of embodiment 26, wherein the reassortant influenza virus grows toa titer of at least about 9 log10 PFU/ml in embryonated eggs. 40 Animmunogenic composition comprising the reassortant influenza virus ofany of embodiments 26 to 39. 41 An influenza vaccine comprising thereassortant influenza virus of any of embodiments 26 to 39. 42 A live,cold-adapted, temperature-sensitive, attenuated influenza vaccinecomprising the reassortant influenza virus of any of embodiments 26 to39. 43 The reassortant influenza virus of any of embodiments 26 to 39,wherein the reassortant influenza virus is cold-adapted, temperaturesensitive, or attenuated. 44 A reassortant influenza virus, comprisinggenomic segments 1, 2, 3, 5, and 8 from influenza strain MDV-A, genomicsegment 7 from influenza strain A/Puerto Rico/8/34 (A/PR/8/34), andgenomic segments 4 and 6 from an influenza strain. 45 The reassortantinfluenza virus of embodiment 44, wherein the reassortant influenzavirus grows to a titer of at least about 7.5 log10 PFU/ml in embryonatedeggs. 46 The reassortant influenza virus of embodiment 44, wherein thereassortant influenza virus grows to a titer of at least about 8 log10PFU/ml in embryonated eggs. 47 The reassortant influenza virus ofembodiment 44, wherein the reassortant influenza virus grows to a titerof at least about 8.5 log10 PFU/ml in embryonated eggs. 48 Thereassortant influenza virus of embodiment 44, wherein the reassortantinfluenza virus grows to a titer of at least about 9 log10 PFU/ml inembryonated eggs. 49 An immunogenic composition comprising thereassortant influenza virus of embodiment 44. 50 An influenza vaccinecomprising the reassortant influenza virus of embodiment 44. 51 A live,cold-adapted, temperature-sensitive, attenuated influenza vaccinecomprising the reassortant influenza virus of embodiment 44. 52 Anexpression vector comprising a nucleic acid sequence operably linked toa pol I promoter, wherein the nucleic acid sequence encodes an influenzaM1 protein comprising lysine (K) at position 95, alanine (A) at position143, phenylalanine (F) at position 144, or isoleucine (I) at position218, wherein the positions correspond to the M1 protein of influenzastrain MDV-A, with the proviso that the sequence does not encode awild-type M1 protein of influenza strain A/PR/8/34. 53 The expressionvector of embodiment 52, wherein the M1 protein comprises lysine (K) atposition 95, alanine (A) at position 143, phenylalanine (F) at position144, and isoleucine (I) at position 218. 54 An isolated negative senseRNA expressed from the expression vector of embodiment 52 or 53. 55 Aninfluenza M1 protein comprising lysine (K) at position 95, alanine (A)at position 143, phenylalanine (F) at position 144, or isoleucine (I) atposition 218, wherein the positions correspond to the M1 protein ofinfluenza strain MDV-A, with the proviso that the protein is not awild-type M1 protein of influenza strain A/PR/8/34. 56 The influenza M1protein of embodiment 55, comprising lysine (K) at position 95, alanine(A) at position 143, phenylalanine (F) at position 144, and isoleucine(I) at position 218.

7. EXAMPLES

The following examples serve merely to illustrate the invention and arenot intended to limit the invention in any way. Examples 1 and 2, below,demonstrate the identification and characterization of M1 amino acidsthat affect influenza virus replication.

7.1 Example 1 Identification of M1 Amino Acids that Affect InfluenzaVirus Replication Capacity

This example describes identification of amino acid residues ininfluenza viral protein M1 that affect replication capacity inembryonated hens' eggs. Previous studies had identified a correlationbetween M gene genotype and increased viral replication capacity. SeeKlimov et al., 1991, Virus Res. 19:105-114. For example, influenza Astrain A/PR/8/34 replicates to a titer (1×10^(8.76) PFU/ml) more than10-fold greater than strain MDV-A (1×10^(7.35) PFU/ml) in embryonatedhens' eggs. See FIG. 1.

Accordingly, the sequences of A/PR/8/34 and MDV-A M1 and M2 proteinswere aligned and compared to identify amino acids that differ betweenthe two strains. The sequence comparison identified six amino aciddifferences in the M1 protein (see FIG. 2) and 12 amino acid differencesin the M2 protein (data not shown).

7.2 Example 2 Replication Capacity of Recombinant and/or ReassortantInfluenza Virus Comprising M1 Mutations

This example describes experiments to identify and characterize theeffects of the variability in the M1 protein on influenza virusreplication capacity. Methods for making reassortant influenza viruses,titering influenza viruses, infecting hens' eggs with influenza viruses,and rescue of influenza viruses from vectors are described, for example,in U.S. Patent Application Publication Nos. US20050158342,US20040029251, and US20050266026, and in Hoffman et al., 2002, Proc.Nat. Acad. Sci. USA 99:11411-6; Jin et al., 2003, Virology 306:18-24;and in Chen et al., 2006, Virology 345:416-23, each of which is herebyincorporated by reference.

Site directed mutagenesis of single amino acids and combinations ofamino acids was performed to identify the effects of these amino aciddifferences on viral replication capacity of a cold adapted, temperaturesensitive, attenuated A/Wyoming/3/03 strain. This strain was made from a6:2 reassortant using the backbone of MDV-A and the HA and NA encodingsegments from A/Wyoming/3/03, and is referred to herein as ca at tsA/Wyoming/3/03.

In these experiments, the amino acids at positions 41, 95, 116, 143,144, 218 and various combinations thereof were mutated from the aminoacid found in the A/Wyoming/3/03 M1 protein to the amino acid found inthe A/PR/8/34 M1 protein. For controls, the ca at ts A/Wyoming/3/03 6:2reassortant virus, a 5:3 reassortant virus containing the MDV-A backboneexcept for the M segment and M, HA and NA from A/Wyoming/3/03, and a 5:3reassortant containing the MDV-A backbone except for the M segment, theM segment from A/PR/8/34, and HA and NA from A/Wyoming/3/03 were used.These various reassortant viruses were used to infect hens' eggs usingconventional techniques and titered with a plaque assay on MDCK cells.Results of these experiments are presented in FIG. 3.

As shown in FIG. 3, the ca at ts A/Wyoming/3/03 reassortant virusexhibited the lowest titer of the reassortant viruses, while the 5:3 caat ts A/Wyoming/3/03 with an M gene from A/PR/8/34 reassortant virusexhibited the highest titer. As also shown in FIG. 3, mutations atpositions 95, 143, 144, and 218 also affected titer of replication.Changing the MDV-A M1 residue 95 from arginine to lysine slightlyincreased replication capacity; changing the MDV-A M1 residue 218 fromvaline to threonine slightly increased replication capacity; changingthe MDV-A M1 residues 143 and 144 from valine and leucine to alanine andphenylalanine, respectively, slightly lowered replication capacity;changing the MDV-A M1 residues 143, 144, and 218 from valine, leucine,and valine to alanine, phenylalanine, and threonine, respectively,slightly increased replication capacity; and changing the MDV-A M1residues 95, 143, 144, and 218 from arginine, valine, leucine, andvaline to lysine, alanine, phenylalanine, and threonine, respectively,greatly increased replication capacity.

To confirm these effects, additional site-directed mutants wereconstructed altering the residues of the A/PR/8/34 M1 protein to thosefound in the MDV-A M1 protein. Vectors encoding these M gene mutantswere used to produce 5:3 reassortant viruses with the MDV-A backbone,the ca A/Wyoming/3/03 HA and NA, and the appropriately modifiedA/PR/8/34 M gene. The various reassortant viruses were introduced intohens' eggs and titered by plaque assay on MDCK cells as described above.Viral titers obtained from these site-directed mutants are shown as FIG.4.

As shown in FIG. 4, altering any of positions 65, 143, 144, or 218 fromthe amino acid found in A/PR/8/34 to the amino acid found in MDV-Aresulted in reduced viral replication capacity in hens' eggs. Thesmallest reduction in replication capacity was observed for the singlechange of position 218 from threonine to valine, while the greatestreduction was observed for the double change of positions 143 and 144from alanine and phenylalanine to valine and leucine, respectively.

Whereas, particular embodiments of the invention have been describedabove for purposes of description, it will be appreciated by thoseskilled in the art that numerous variations of the details may be madewithout departing from the invention as described in the appendedclaims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

1. A method for increasing the replication capacity of an influenzavirus, comprising altering an at least one amino acid of the influenzavirus M1 protein, wherein the at least one amino acid is at a positioncorresponding to at least one position 95, 143, 144 or 218 of the M1protein of influenza strain MDV-A as shown in SEQ ID NO:1; wherein thealtering at the position corresponding to position 95 is to a lysine;wherein the altering at the position corresponding to position 143 is toan alanine; wherein the altering at the position corresponding toposition 144 is to a phenylalanine; or wherein the altering at theposition corresponding to position 218 is to an isoleucine; and whereinthe altering increases thereby increasing the replication capacity ofthe influenza virus.
 2. The method of claim 1, wherein at least two ofthe amino acids at the positions corresponding to positions 95, 143,144, or 218 are altered.
 3. The method of claim 1, wherein at leastthree of the amino acids at the positions corresponding to positions 95,143, 144, or 218 are altered.
 4. The method of claim 1, wherein the atleast one amino acid is at the position corresponding to position
 95. 5.(canceled)
 6. The method of claim 1, wherein the at least one amino acidis at the position corresponding to position
 143. 7. (canceled)
 8. Themethod of claim 1, wherein the at least one amino acid is at theposition corresponding to position
 144. 9. (canceled)
 10. The method ofclaim 1, wherein the at least one amino acid is at the positioncorresponding to position
 218. 11. (canceled)
 12. The method of claim 12wherein the altering is of amino acids at positions corresponding topositions 95, 143, 144 and
 218. 13-17. (canceled)
 18. The method ofclaim 1, wherein the replication capacity of the influenza virus isincreased at least 2-fold relative to the same influenza virus in theabsence of the alteration. 19-20. (canceled)
 21. The method of claim 1,wherein the replication capacity of the influenza virus is increased atleast 10-fold relative to the same influenza virus in the absence of thealteration.
 22. The method of claim 1, wherein the influenza virus growsto a titer of at least about 7.5 log₁₀ PFU/ml in embryonated eggs. 23.The method of claim 1, wherein the influenza virus grows to a titer ofat least about 8 log₁₀ PFU/ml in embryonated eggs. 24-25. (canceled) 26.A reassortant influenza virus comprising an M1 protein, wherein the M1protein comprises an alteration in at least one amino acid at a positioncorresponding to position 95, 143, 144, or 218 of the M1 protein ofinfluenza strain MDV-A as shown in SEQ ID NO: 1 wherein the alterationof the position corresponding to position 95 is to lysine, whereinalteration at the position corresponding to position 143 is to alanine,wherein alteration at the position corresponding to position 144 is tophenylalanine, and wherein alteration at the position corresponding toposition 218 is to isoleucine.
 27. The reassortant influenza virus ofclaim 26, wherein the alteration is at the position corresponding toposition
 95. 28. The reassortant influenza virus of claim 26, whereinthe alteration is at the position corresponding to position
 143. 29. Thereassortant influenza virus of claim 26, wherein the alteration is atthe position corresponding to position
 144. 30. The reassortantinfluenza virus of claim 26, wherein the alteration is at the positioncorresponding to position
 218. 31. The reassortant influenza virus ofclaim 27, further comprising the alteration at the positioncorresponding to position 143, and the alteration at position 144, andthe alteration at the position corresponding to position
 218. 32-39.(canceled)
 40. An immunogenic composition comprising the reassortantinfluenza virus of claim
 26. 41. An influenza vaccine comprising thereassortant influenza virus of claim
 26. 42-56. (canceled)